TWI487535B - Toll like receptor 3 antagonists, methods and uses - Google Patents
Toll like receptor 3 antagonists, methods and uses Download PDFInfo
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- TWI487535B TWI487535B TW094141961A TW94141961A TWI487535B TW I487535 B TWI487535 B TW I487535B TW 094141961 A TW094141961 A TW 094141961A TW 94141961 A TW94141961 A TW 94141961A TW I487535 B TWI487535 B TW I487535B
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Description
本申請案請求2004年11月30日申請的美國臨時申請案60/631,815、2004年12月15日申請的美國臨時申請案60/636,399、2005年1月6日申請的美國臨時申請案60/641,877、2005年8月31日申請的美國臨時申請案60/713,195及2005年10月18日申請的美國臨時申請案60/727,610,其全部內容併入本文以資參考。This application claims US Provisional Application No. 60/631,815 filed on November 30, 2004, U.S. Provisional Application No. 60/636,399 filed on December 15, 2004, and US Provisional Application No. 60/ filed on January 6, 2005 641, 877, U.S. Provisional Application No. 60/713, 195, filed on Aug. 31, 2005, and U.S. Provisional Application Serial No. 60/727, 610, filed on
本發明乃有關類鐸受體3(TLR3)拮抗劑、編碼TLR3拮抗劑之多核苷酸或其片段、及彼等之製法及用途。The present invention relates to a terpenoid receptor 3 (TLR3) antagonist, a polynucleotide encoding a TLR3 antagonist or a fragment thereof, and methods of making and using same.
與炎性疾病有關聯之病變代表保健上受到重大挑戰,有時會痛苦、衰弱及致命。舉例而言,在美國,敗血症及與敗血症相關的疾病每年影響超過750,000人,其致死率為28-50%,每年造成215,000人死亡(Natansonet al.,Crit.Care Med.26 :1927-1931(1998);Anguset al.,Crit.Care Med.29 :1303-1310(2001))。其他炎性疾病例如炎性腸道疾病(IBD)、克隆氏症(Crohn’s disease)與潰瘍性結腸炎每年於美國影響超過1百萬人(Hanaueret al.,Rev.Gastroenterol.Disord.3 :81-92(2003))。Lesions associated with inflammatory diseases represent a major challenge in health care, sometimes painful, debilitating and fatal. For example, in the United States, sepsis and sepsis-related diseases affect more than 750,000 people each year, with a mortality rate of 28-50%, resulting in 215,000 deaths per year (Natanson et al., Crit. Care Med. 26 :1927-1931) (1998); Angus et al., Crit. Care Med. 29 : 1303-1310 (2001)). Other inflammatory diseases such as inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis affect more than 1 million people each year in the United States (Hanauer et al., Rev. Gastroenterol. Disord. 3:81 -92 (2003)).
影響肺功能之炎性肺部疾病例如慢性阻塞性肺疾(COPD)、氣喘與肺感染在美國亦影響相當數量的人民,例如,COPD,據估計影響約1千萬美國人,且流行率正上升中(Mapelet al.,Manag.Care Interface 17 :61-66(2004))。與彼等炎性疾病相關之病變及彼等疾病之惡化對於健康與經濟具有重大之衝擊。Inflammatory lung diseases affecting lung function such as chronic obstructive pulmonary disease (COPD), asthma and lung infections also affect a significant number of people in the United States, for example, COPD, which is estimated to affect approximately 10 million Americans, and the prevalence rate is positive Rising (Mapel et al., Manag. Care Interface 17 : 61-66 (2004)). The lesions associated with their inflammatory diseases and the deterioration of their diseases have a major impact on health and the economy.
肺部疾病例如氣喘與COPD之惡化,其特徵為症狀加劇及肺功能衰退。病毒感染與許多肺部疾病相關聯(Johnston,Am.J.Respir.Crit.Care Med.152 :S46-52(1995);Bandiet al,FEMS Immunol.Med.Microbiol.37 :69-75(2003)),一般相信其為惡化之主要原因。跟隨病毒感染之後,肺部中促炎性細胞介素之分泌代表促成多種肺部疾病的炎性反應之決定性步驟(Gemet al.,Am.J.Respir.Cell.Mol.Biol.28 :731-737(2003);Panina-Bordignonet al.,Curr.Opin.Pulm.Med.9 :104-110(2003))。Pulmonary diseases such as asthma and worsening of COPD are characterized by increased symptoms and decreased lung function. Viral infection is associated with many lung diseases (Johnston, Am. J. Respir . Crit. Care Med. 152 : S46-52 (1995); Bandi et al, FEMS Immunol. Med . Microbiol. 37: 69-75 (2003) )), generally believed to be the main cause of deterioration. Following viral infection, the secretion of pro-inflammatory interleukins in the lungs represents a decisive step in the inflammatory response that contributes to a variety of lung diseases (Gem et al., Am. J. Respir. Cell . Mol. Biol. 28 : 731). -737 (2003); Panina-Bordignon et al., Curr. Opin. Pulm. Med . 9 : 104-110 (2003)).
再者,胰島素抗阻已被認定係所謂代謝症候群之必要特徵,其包括葡萄糖耐受不良、胰島素抗阻、肥胖、高三酸甘油酯血症、低HDL膽固醇、高血壓、及加速型動脈硬化症(Wisse,J.Am.Soc.Nephrol.15 :2792-20 800(2004))。雖然2型糖尿病與胰島素抗阻間之傾向已清楚地被確立,控制胰島素抗阻及2型糖尿病之分子與細胞機制仍很模糊。Furthermore, insulin resistance has been identified as a necessary feature of the so-called metabolic syndrome, including glucose intolerance, insulin resistance, obesity, hypertriglyceridemia, low HDL cholesterol, hypertension, and accelerated atherosclerosis. (Wisse, J. Am . Soc . Nephrol . 15: 2792-20 800 (2004)). Although the tendency between type 2 diabetes and insulin resistance has been clearly established, the molecular and cellular mechanisms controlling insulin resistance and type 2 diabetes are still blurred.
肥胖個體促炎性細胞介素(例如TNF-α、IL-1b與IL-6)含量升高之事實已促成由肥胖誘發之胰島素抗阻係炎性疾病之假說(Karinet al.,Nat.Rev.Drug Discov.3 :17-26(2004))。因此,炎症、肥胖、胰島素抗阻及異常脂質代謝可能構成代謝症候群之常見特徵。事實上,可能干擾炎性轉錄因子(例如NF-kβ與IKKβ)之非類固醇藥物,例如環氧化酶抑制劑,增加2型糖尿病動物模式與人類病患之胰島素敏感性(Karinet al. ,文獻同前)。此外,由處於IKKb狀態的基因剔除小鼠於髓樣細胞中表現總體胰島素敏感性且成為免於胰島素抗阻之能力顯示,新近數據為胰島素抗阻與炎症間之關係提供支撐(Arkanet al.,Nat.Med.11 :191-198(2005);Caiet al.,Nat.Med. 11:183-90(2005))。總之,彼等結果為肥胖、胰島素抗阻及2型糖尿病與炎性疾病之間的關聯提供強有力之基本原由。The fact that elevated levels of pro-inflammatory interleukins (e.g., TNF-[alpha], IL-Ib, and IL-6) in obese individuals has contributed to the hypothesis of obesity-induced insulin resistance inflammatory diseases (Karin et al., Nat. Rev. Drug Discov. 3 : 17-26 (2004)). Therefore, inflammation, obesity, insulin resistance, and abnormal lipid metabolism may constitute a common feature of metabolic syndrome. In fact, non-steroidal drugs that may interfere with inflammatory transcription factors (such as NF-kβ and IKKβ), such as cyclooxygenase inhibitors, increase the insulin sensitivity of animal models of type 2 diabetes and human patients (Karin et al. , literature Cit). In addition, gene knockout mice in the IKKb state show overall insulin sensitivity in myeloid cells and show immunity from insulin resistance, and recent data provide support for the relationship between insulin resistance and inflammation (Arkan et al. , Nat . Med. 11: 191-198 (2005); Cai et al., Nat. Med. 11: 183-90 (2005)). In conclusion, their results provide a strong rationale for the association between obesity, insulin resistance, and type 2 diabetes and inflammatory diseases.
宿主免疫系統對微生物抗原之識別係透過固有免疫受體而傳介,其活化作用代表開始炎性反應之重要步驟。類鐸受體(TLR)代表與生俱來之免疫受體家族,於傳介對外來抗原之免疫反應中扮演決定性角色。舉例而言,TLR3為一種哺乳動物模式識別受體,可識別雙股(ds)RNA以及合成用雙股RNA類似物多-核糖肌苷酸-核糖胞苷酸(多(I:C))(Alexopoulouet al.,Nature 413 :732-238(2001))。此外,已證實TLR3可識別內源配位體(例如自壞死細胞釋放之mRNA)(Karikoet al.,J.Biol.Chem.26 :12542-12550(2004),暗示於發炎部位之壞死細胞死亡可能促成TLR3之活化。The recognition of microbial antigens by the host immune system is transmitted through innate immune receptors, and its activation represents an important step in initiating an inflammatory response. Terpenoid receptors (TLRs) represent a family of immune receptors that play a decisive role in the immune response to foreign antigens. For example, TLR3 is a mammalian pattern recognition receptor that recognizes double-stranded (ds) RNA and synthetic double-stranded RNA analog poly-ribo-inosinic-ribonucleotide (poly(I:C)) ( Alexopoulou et al., Nature 413 : 732-238 (2001)). Furthermore, TLR3 has been shown to recognize endogenous ligands (eg, mRNA released from necrotic cells) (Kariko et al., J. Biol. Chem . 26: 12542-12550 (2004), suggesting necrotic cell death at the site of inflammation. May promote the activation of TLR3.
利用多(I:C)或利用內源mRNA配位體之TLR3活化作用誘發促炎性細胞介素與化學激素之分泌,此發現暗示TLR3促效劑於與感染相關之炎症期間調控疾病後果。因此,活體內之TLR3接合作用被認為係鑑於病毒感染(Tabetaet al.,Proc.Nati.Acad.Sci.USA 101 :3516-3521(2004))或與發炎相關之壞死(Karikoet al.,J.Bid.Chem.26 :12542-12550(2004))而發生。整體而言,彼等資訊說明TLR3之接合觸發磷酸化作用及轉錄活化結果等級聯反應,導使產生被認為係促成固有免疫力的許多炎性細胞介素(綜述於Takeda and Akira,J.Derm.Sci.34 :73-82(2004))。再者,彼等資訊暗示持續之TLR3活化作用可能係調控與炎性疾病相關的感染之關鍵成分。已公告之資訊證實,促炎性細胞介素之大量表現與全身性發炎反應症候群、與感染相關的急性細胞介素發作(綜述於Van Amersfoortet al.,Clin.Microbiol.Rev.16 :379-414(2003))、由免疫傳介之慢性疾病例如類風濕性關節炎(綜述於Miossecet al.,Curr.Opin.Rheumatol.16 :218-222(2004))及炎性腸道疾病(綜述於Ogata and Hibi,Curr.Pharm.Des.9 :1107-1113(2003))相關,彼等表現提供前述假說之支撐。The secretion of pro-inflammatory interleukins and chemical hormones is induced by multiple (I:C) or TLR3 activation using endogenous mRNA ligands. This finding suggests that TLR3 agonists regulate disease outcomes during infection-associated inflammation. Therefore, TLR3 zygosity in vivo is thought to be due to viral infection (Tabeta et al., Proc. Nati . Acad. Sci. USA 101 : 3516-3521 (2004)) or inflammation-related necrosis (Kariko et al., J. Bid . Chem. 26 : 12542-12550 (2004)). Overall, their information indicates that TLR3 junction triggers a cascade of phosphorylation and transcriptional activation results, leading to the production of many inflammatory interleukins that are thought to contribute to innate immunity (reviewed in Takeda and Akira, J.Derm). .Sci. 34 : 73-82 (2004)). Furthermore, their information suggests that sustained TLR3 activation may be a key component in the regulation of infections associated with inflammatory diseases. The published information confirms that a large number of pro-inflammatory interleukins are associated with systemic inflammatory response syndrome and acute interleukin seizures associated with infection (reviewed in Van Amersfoort et al., Clin. Microbiol. Rev. 16 :379- 414 (2003)), chronic diseases induced by immunization such as rheumatoid arthritis (reviewed in Miossec et al., Curr. Opin . Rheumatol . 16: 218-222 (2004)) and inflammatory bowel disease (review) in Ogata and Hibi, Curr.Pharm.Des.9: 1107-1113 ( 2003)) related to their performance of providing support previous hypotheses.
雖然試管內研究已證明刺激具有多(I:C)之肺上皮細胞引起多種細胞介素、化學激素之分泌且誘發轉錄因子及使TLRs增加表現(Iekiet al.,Clin.Exp.Allergy 34 :745-52(2004);Shaet al.,Am.J.Respir.Cell.Mol.Biol.31 :358-64(2004)),然而此等事件之生理關聯性仍不清楚。Although in vitro studies have demonstrated that stimulation of multiple (I:C) lung epithelial cells causes secretion of various interleukins, chemical hormones and induces transcription factors and increases TLRs (Ieki et al., Clin. Exp . Allergy 34 : 745-52 (2004); Sha et al., Am. J. Respir. Cell . Mol. Biol. 31 :358-64 (2004)), however, the physiological relevance of these events remains unclear.
與炎性疾病及其他(例如與感染有關聯者)相關之彼等病變對於健康與經濟具有重大之衝擊。然而,儘管許多醫藥領域已有進步,適用於許多彼等疾病之處理選擇與治療相對地極少。These lesions associated with inflammatory diseases and other (eg, those associated with infection) have a significant impact on health and the economy. However, despite the advances in many areas of medicine, treatment options and treatments for many of these diseases are relatively rare.
例如,使用高劑量皮質類固醇與抗-IgE(例如XOLAIR廠牌之奧馬佐單抗(omalizumab))處理肺部疾病之惡化;已證實吸入性皮質類固醇組合β2促效劑對於減少惡化之發生具有效力。然而,由於彼等療法僅降低惡化進展之風險且與重大的副作用相關,因此業界對於預防及治療肺部疾病惡化之替代治療方法有所需求。For example, use high doses of corticosteroids with anti-IgE (eg XOLAIR The label of omalizumab (omalizumab) treats the deterioration of lung disease; the inhaled corticosteroid combination β2 agonist has been shown to be effective in reducing the onset of exacerbations. However, as their therapies only reduce the risk of worsening progression and are associated with significant side effects, there is a need in the industry for alternative treatments to prevent and treat the progression of lung disease.
因此,存在瞭解TLR3於炎性疾病上的角色及開拓此角色以開發有效治療彼等疾病之製劑(例如拮抗劑)之需求。Therefore, there is a need to understand the role of TLR3 in inflammatory diseases and to develop this role to develop formulations (e.g., antagonists) that are effective in treating such diseases.
本發明之一態樣為抑制選自包括IL-6、IL-8與MIP1-α的細胞介素之細胞生產之類鐸受體3(TLR3)拮抗劑。One aspect of the invention is the inhibition of a steroid receptor 3 (TLR3) antagonist produced from a cell selected from the group consisting of IL-6, IL-8 and MIP1-α.
本發明之一態樣為與TLR3具反應性之單離抗體,其具有單株抗體之抗原結合能力,含有如SEQ ID NOs:9、11與13所示之重鏈互補決定區(CDRs)之胺基酸序列及如SEQ ID NOs:19、21與23所示之輕鏈CDRs之胺基酸序列。One aspect of the invention is an isolated antibody reactive with TLR3 having the antigen binding ability of a monoclonal antibody comprising heavy chain complementarity determining regions (CDRs) as set forth in SEQ ID NOs: 9, 11 and 13. Amino acid sequence and amino acid sequence of light chain CDRs as set forth in SEQ ID NOs: 19, 21 and 23.
本發明之另一態樣為與TLR3具反應性之單離抗體,其含有如SEQ ID NOs:9、11與13所示之重鏈互補決定區(CDRs)之胺基酸序列及如SEQ ID NOs:19、21與23所示之輕鏈CDRs之胺基酸序列。Another aspect of the invention is an isolated antibody reactive with TLR3 comprising an amino acid sequence of heavy chain complementarity determining regions (CDRs) as set forth in SEQ ID NOs: 9, 11 and 13, and SEQ ID NOs: amino acid sequences of the light chain CDRs shown in 19, 21 and 23.
本發明之另一態樣為一種單離抗體,其具有如式(I)所示之VH CDR1胺基酸序列:Thr Thr Tyr Trp Xaa1 His(I)式中Xaa1 為Ile或Met(SEQ ID NO:61);如式(II)所示之VH CDR2胺基酸序列:Glu Ile Asn Pro Asn Asn Gly Arg Ile Asn Xaa2 Xaa3 Glu Lys Xaa4 Lys Thr(II)式中Xaa2 為Tyr或Gly,Xaa3 為Asn或Ala及Xaa4 為Phe或Gly(SEQ ID NO:62);及如式(III)所示之VH CDR3胺基酸序列:Val Gly Val Xaa5 Ile Thr Thr Phe Pro Tyr(III)式中Xaa5 為Met或Ile(SEQ ID NO:63);及具有如SEQ ID NOs:19、21與23所示之胺基酸序列之VL CDRs。Another aspect of the invention is an isolated antibody having a VH CDR1 amino acid sequence as shown in formula (I): Thr Thr Tyr Trp Xaa 1 His (I) wherein Xaa 1 is Ile or Met ( SEQ ID NO: 61); V H CDR2 amino acid sequence as shown in formula (II): Glu Ile Asn Pro Asn Asn Gly Arg Ile Asn Xaa 2 Xaa 3 Glu Lys Xaa 4 Lys Thr (II) where Xaa 2 Is Tyr or Gly, Xaa 3 is Asn or Ala and Xaa 4 is Phe or Gly (SEQ ID NO: 62); and V H CDR3 amino acid sequence as shown in formula (III): Val Gly Val Xaa 5 Ile Thr Thr Phe Pro Tyr (III) wherein Xaa 5 is Met or Ile (SEQ ID NO: 63); and VL CDRs having amino acid sequences as shown in SEQ ID NOs: 19, 21 and 23.
本發明之另一態樣為編碼含有如SEQ ID NOs:9、11與13所示之CDR胺基酸序列之抗體重鏈之單離多核苷酸。Another aspect of the invention is an isolated polynucleotide encoding an antibody heavy chain comprising a CDR amino acid sequence as set forth in SEQ ID NOs: 9, 11 and 13.
本發明之另一態樣為編碼含有如SEQ ID NOs:19、21與23所示之CDR胺基酸序列之抗體輕鏈之單離多核苷酸。Another aspect of the invention is an isolated polynucleotide encoding an antibody light chain comprising a CDR amino acid sequence as set forth in SEQ ID NOs: 19, 21 and 23.
本發明之另一態樣為編碼含有如SEQ ID NOs:6、25、27、29、31、45、47、49、51或53所示之胺基酸序列之抗體重鏈之單離多核苷酸。Another aspect of the invention is an isolated polynucleoside encoding an antibody heavy chain comprising an amino acid sequence as set forth in SEQ ID NOs: 6, 25, 27, 29, 31, 45, 47, 49, 51 or 53 acid.
本發明之另一態樣為編碼含有如SEQ ID NOs:16、33、35、37或39所示之胺基酸序列之抗體輕鏈之單離多核苷酸。Another aspect of the invention is an isolated polynucleotide encoding an antibody light chain comprising an amino acid sequence as set forth in SEQ ID NOs: 16, 33, 35, 37 or 39.
本發明之另一態樣為治療或預防炎性疾病之方法,該方法包括投與有其需要之病患預防有效量之TLR3拮抗劑足以治療或預防該炎性疾病之時間。Another aspect of the invention is a method of treating or preventing an inflammatory disease, the method comprising administering to a patient in need thereof a prophylactically effective amount of a TLR3 antagonist sufficient to treat or prevent the inflammatory condition.
本發明之另一態樣為增加細胞增殖率之方法,該方法包括使TLR3拮抗劑與表現TLR3受體之細胞接觸足以增加該細胞增殖率之時間。Another aspect of the invention is a method of increasing the rate of cell proliferation, the method comprising contacting a TLR3 antagonist with a cell expressing a TLR3 receptor for a time sufficient to increase the rate of proliferation of the cell.
本說明書中引用之所有公告案,包括惟不限於專利案及專利申請案,均併入本文以資參考。All publications cited in this specification, including but not limited to patents and patent applications, are hereby incorporated by reference.
本文所用之「拮抗劑」一詞意指利用任何機制部分或完全抑制另一分子(例如受體)之效力之分子。本文所用之「TLR3拮抗劑」或「與TLR3具反應性」之化合物係敘述能直接或間接,實質上抵消、減少或抑制TLR3生物活性或TLR3受體活化作用之分子。此等拮抗劑可為,例如,小有機分子、胜肽、多肽、融合蛋白、抗體、抗體片段、模擬體或多核苷酸。The term "antagonist" as used herein, refers to a molecule that utilizes any mechanism to partially or completely inhibit the potency of another molecule, such as a receptor. As used herein, "TLR3 antagonist" or "reactive with TLR3" describes a molecule that directly or indirectly substantially counteracts, reduces or inhibits TLR3 biological activity or TLR3 receptor activation. Such antagonists can be, for example, small organic molecules, peptides, polypeptides, fusion proteins, antibodies, antibody fragments, mimetics or polynucleotides.
本文所用之「抗體」一詞廣義上意指及包含免疫球蛋白或抗體分子包括多株抗體、單株抗體包括鼠類、人類、人類適應、人類化與嵌合型單株抗體及抗體片段。As used herein, the term "antibody" is used broadly to mean and encompass immunoglobulin or antibody molecules, including polyclonal antibodies, monoclonal antibodies including murine, human, human adapted, humanized and chimeric monoclonal antibodies and antibody fragments.
一般而言,抗體係展現與專一抗原之結合專一性之蛋白質或胜肽。完整之抗體為雜四聚體醣蛋白,由兩個完全相同的輕鏈與兩個完全相同的重鏈組成。典型地,各輕鏈藉由一個共價二硫鍵與重鏈連接;不同的同功型免疫球蛋白之重鏈,其二硫鍵個數也不同。各重鏈與輕鏈亦具有規則相間之鏈間二硫橋鍵。各重鏈於一端具有可變區(VH ),其後為一些恒定區。各輕鏈於一端具有可變區(VL ),另一端具有恒定區;輕鏈之恒定區與重鏈之第一恒定區對齊,輕鏈可變區與重鏈之可變區對齊。任一種脊椎動物之諸抗體輕鏈根據其恒定區之胺基酸序列,可歸入兩個清楚區隔類型之一者,亦即κ或λ。In general, an anti-system exhibits a protein or peptide that binds to a specific antigen. The intact antibody is a heterotetrameric glycoprotein consisting of two identical light chains and two identical heavy chains. Typically, each light chain is linked to a heavy chain by a covalent disulfide bond; the heavy chain of a different isoform immunoglobulin has a different number of disulfide bonds. Each heavy chain and light chain also has a regular interchain disulfide bridge. Each heavy chain has a variable region ( VH ) at one end followed by some constant regions. Each light chain has at one end a variable region (V L), the other end having a constant region; the first constant domain of the light chain is aligned with the heavy chain, the variable region of the light chain variable region of the heavy chains are aligned. The antibody light chain of any of the vertebrate can be classified into one of two distinct compartment types, namely kappa or lambda, depending on the amino acid sequence of its constant region.
免疫球蛋白視其重鏈恒定區胺基酸序列而定,可分為五個主要種類,亦即IgA、IgD、IgE、IgG與IgM。IgA與IgG再進一步細分為IgA1 、IgA2 、IgG1 、IgG2 、IgG3 與IgG4 等同功型。Immunoglobulins are classified into five major classes depending on their heavy chain constant region amino acid sequence, namely IgA, IgD, IgE, IgG and IgM. IgA and IgG are further subdivided into IgA 1 , IgA 2 , IgG 1 , IgG 2 , IgG 3 and IgG 4 equivalents.
「抗體片段」一詞意指部分完整抗體,通常為完整抗體之抗原結合或可變區。抗體片段之實例包含Fab、Fab’、F(ab’)2 與諸Fv片段、雙功能抗體(diabodies)、單鏈抗體分子及由至少兩個完整抗體形成之多專一性抗體。The term "antibody fragment" means a partially intact antibody, usually the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments, diabodies, single-chain antibody molecules, and multi-specific antibodies formed from at least two intact antibodies.
本文所用之「抗原」一詞意指具有直接或間接產生抗體能力之任何分子。「抗原」之界定內尚包含編碼蛋白質之核酸。As used herein, the term "antigen" means any molecule that has the ability to produce antibodies directly or indirectly. The nucleic acid encoding the protein is also included in the definition of "antigen".
「CDRs」係界定為抗體之互補決定區胺基酸序列,彼等為免疫球蛋白重鏈與輕鏈之高可變區。參閱,例如,Kabatet al., Sequences of Proteins of Immunological Interest,4th ed.,U.S.Department of Health and Human Services,National Institutes of Health(1987)。免疫球蛋白可變部分中有三個重鏈及三個輕鏈CDRs或CDR區域。因此,本文所用之「CDRs」係指所有三個重鏈CDR、或所有三個輕鏈CDRs,或適當時,則為所有重鏈以及所有輕鏈。"CDRs" are defined as the amino acid sequence of the complementarity determining region of an antibody, which are hypervariable regions of immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDepartment of Health and Human Services, National Institutes of Health (1987). There are three heavy chains and three light chain CDRs or CDR regions in the immunoglobulin variable portion. Thus, "CDRs" as used herein refers to all three heavy chain CDRs, or all three light chain CDRs, or, where appropriate, all heavy chains and all light chains.
CDRs提供抗體結合於抗原或抗原決定部位之多數接觸殘基。本發明所關注之CDRS係衍生自供者抗體可變重鏈與輕鏈序列,其包含天然存在的CDRs之類似物,該類似物亦共有或保留與彼等所從出的供者抗體相同之抗原結合專一性及/或中和能力。CDRs provide for the majority of contact residues of an antibody that binds to an antigen or epitope. The CDRSs of interest in the present invention are derived from donor antibody variable heavy and light chain sequences comprising analogs of naturally occurring CDRs which also share or retain the same antigen as the donor antibody from which they are derived. Combine specificity and/or neutrality.
本文所用之「上皮細胞」一詞意指源自動物之覆蓋部分游離表面(例如,皮膚)或填充管或腔(例如,結腸)的膜狀細胞組織之細胞。此等細胞可予以單離或包含部分或更高度有系統之細胞組群,例如見於組織、器官或彼等之試管內模式者。As used herein, the term "epithelial cells" means cells derived from the free surface of an animal covering a portion of the surface (eg, the skin) or the membranous tissue of a tube or lumen (eg, the colon). Such cells may be isolated or comprise a portion or a more highly systematic group of cells, such as those found in tissues, organs or their in vitro modes.
「同質物」一詞意指與參照序列具有介於40%與100%間之序列同一性之蛋白質序列。hTLR3之同質物包括與已知hTLR3序列具有介於40%與100%間之序列同一性之多肽。兩個胜肽鏈間之同一性百分比可藉使用Vector NTI v.9.0.0(Invitrogen Corp.,Carslbad,CA)AlignX模數預設設定值之成對排比法予以測定。「TLR3」意指hTLR3及其同質物。全長TLR3胺基酸序列及編碼用多核苷酸序列分別示於SEQ ID NOs:1與2。The term "homomorphism" means a protein sequence having a sequence identity between 40% and 100% with respect to a reference sequence. Homologs of hTLR3 include polypeptides having between 40% and 100% sequence identity to known hTLR3 sequences. The percent identity between the two peptide chains can be determined by pairwise alignment using the Vector NTI v.9.0.0 (Invitrogen Corp., Carslbad, CA) AlignX modulus preset setting. "TLR3" means hTLR3 and its homologues. The full-length TLR3 amino acid sequence and the coding polynucleotide sequence are shown in SEQ ID NOs: 1 and 2, respectively.
本文所用之「組合」一詞意指所述製劑可於混合物中一起,呈單一製劑同時或呈單一製劑以任何順序相繼投與動物。As used herein, the term "combination" means that the preparations can be administered together in a mixture in a single preparation, either simultaneously or in a single preparation, in any order.
本文所用之「炎性疾病」一詞意指部分由細胞介素、化學激素、或炎性細胞(例如嗜中性白血球、單核白血球與淋巴細胞)活性所傳介之對細胞傷害之局部反應,其特徵於多數情形下為疼痛、紅、腫及喪失組織功能。本文所用之「炎性肺部疾病」意指侵襲肺部或與肺部相關聯之炎性疾病。As used herein, the term "inflammatory disease" means a partial response to cellular damage transmitted by interleukins, chemical hormones, or inflammatory cells (eg, neutrophils, mononuclear leukocytes, and lymphocytes). It is characterized by pain, redness, swelling and loss of tissue function in most cases. As used herein, "inflammatory lung disease" means an inflammatory disease that affects the lungs or is associated with the lungs.
本文所用之「模擬體」一詞意指具下式(I)之蛋白質:(V1-Pep-Lk-V2-Hg-CH 2-CH 3)(t)(I)其中V1為免疫球蛋白可變區N端之一部分,Pep為與細胞表面TLR3結合之多肽,Lk為多肽或化學鍵結,V2為免疫球蛋白可變區C端之一部分,Hg為免疫球蛋白鉸合區域之一部分,CH 2為免疫球蛋白重鏈CH 2恒定區,CH 3為免疫球蛋白重鏈CH 3恒定區及t為1至10之整數。模擬體視構築體中存在的重鏈恒定功能部位胺基酸序列而定,可模擬不同種類的免疫球蛋白分子(例如IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgD與IgE)之性質與功能。於一些模擬體具體實例中,V1可能不存在。本發明模擬體拮抗劑透過與細胞表面TLR3結合而影響TLR3生物活性。The term "mimetic body" as used herein means a protein of the following formula (I): (V1-Pep-Lk-V2-Hg-C H 2-C H 3)(t)(I) wherein V1 is an immunoglobulin One part of the N-terminus of the variable region of the protein, Pep is a polypeptide that binds to the cell surface TLR3, Lk is a polypeptide or chemical bond, V2 is a part of the C-terminus of the immunoglobulin variable region, and Hg is a part of the immunoglobulin hinge region. C H 2 is an immunoglobulin heavy chain C H 2 constant region, C H 3 is an immunoglobulin heavy chain C H 3 constant region and t is an integer of from 1 to 10. The nature of the heavy chain constant functional site amino acid sequence present in the mimetic construct can mimic the properties of different types of immunoglobulin molecules (eg, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, and IgE). Features. In some simulation examples, V1 may not exist. The mimetic antagonists of the invention affect TLR3 biological activity by binding to cell surface TLR3.
本文所用之「單株抗體」(mAb)一詞意指得自一群實質上同源抗體之抗體(或抗體片段)。單株抗體具高度專一性,典型地係針對單一抗原決定子。修飾詞「單株」表示實質上同源之抗體特性及不需要利用任何特定方法產生抗體。舉例而言,鼠類單株抗體可利用Kohleret al.,Nature 256 :495-497(1975)之融合瘤方法製造。含有衍生自供者抗體(典型地為鼠類)之輕鏈與重鏈可變區結合衍生自接受者抗體(典型地為另一種哺乳動物例如人類)之輕鏈與重鏈恒定區之嵌合型單株抗體可利用揭示於美國專利案4,816,567之方法製備。具有衍生自非人類供者免疫球蛋白(典型地為鼠類)之CDRs及該分子剩餘之衍生自免疫球蛋白之部分係衍生自一或多種人類免疫球蛋白之人類適應單株抗體可利用熟習此項技藝人士已知之技術,例如揭示於美國專利案5,225,539中者,予以製備。視需要地,人類適應單株抗體可進一步利用揭示於Queenet al.,Proc.Natl Acad Sci(USA),86 :10029-10032(1989)及Hodgsonet al.,Bio /Technology,9 :421(1991)之技術,藉由併入經修改之架構支撐殘基以保存結合親和性予以修飾。The term "monoclonal antibody" (mAb) as used herein refers to an antibody (or antibody fragment) obtained from a population of substantially homologous antibodies. Individual antibodies are highly specific and are typically directed against a single antigenic determinant. The modifier "single plant" means essentially homologous antibody properties and does not require the production of antibodies by any particular method. For example, murine monoclonal antibodies can be made using the fusion knob method of Kohler et al., Nature 256 :495-497 (1975). A light chain containing a donor antibody (typically a murine) binds to a heavy chain variable region and is derived from a chimeric version of a light chain and heavy chain constant region derived from a recipient antibody (typically another mammal such as a human) Monoclonal antibodies can be prepared by the method disclosed in U.S. Patent No. 4,816,567. CDRs derived from a non-human donor immunoglobulin (typically a murine) and the remainder of the molecule derived from an immunoglobulin are derived from one or more human immunoglobulins. Techniques known to those skilled in the art, such as those disclosed in U.S. Patent No. 5,225,539, are incorporated herein by reference. As needed, human adaptation to monoclonal antibodies can be further exploited by Queen et al., Proc. Natl Acad Sci (USA), 86 : 10029-10032 (1989) and Hodgson et al., Bio / Technology, 9 :421 ( The technique of 1991) is modified by incorporation of a modified framework to support residues to preserve binding affinity.
用於人類適應化之人類架構序列實例揭示於,例如,www.ncbi.nlm.nih.gov/entrez/query.fcgi;www.ncbi.nih.gov/igblast;www.atcc.org/phage/hdb.html;www.mrc-cpe.cam.ac.uk/ALIGNMENTS.php;www.kabatdatabase.com/top.html;ftp.ncbi.nih.gov/repository/kabat;www.sciquest.com;www.abcam.com;www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/~pedro/research_tools.html;www.whfreeman.com/immunology/CH05/kuby05.htm;www.hhmi.org/grants/lectures/1996/vlab;www.path.cam.ac.uk/~mrc7/mikeimages.html;mcb.harvard.edu/BioLinks/Immunology.html;www.immunologylink.com;pathbox.wustl.edu/~hcenter/index.html;www.appliedbiosystems.com;www.nal.usda.gov/awic/pubs/antibody;www.m.ehim-u.ac.jp/~yasuhito/Elisa.html;www.biodesign.com;www.cancerresearchuk.org;www.biotech.ufl.edu;www.isac-net.org;baserv.uci.kun.nl/~jraats/links1.html;www.recab.uni-hd.de/immuno.bme.nwu.edu;www.mrc~cpe.cam.ac.uk;www.ibt.unam.mx/vir/V_ice.html;http://www.bioinf.org.uk/abs;antibody.bath.ac.uk;www.unizh.ch;www.cryst.bbk.ac.uk/~ubcg07s;www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.html;www.path.cam.ac.uk/~mrc7/humanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stat_aim.html;www.biosci.missouri.edu/smithgp/index.html;www.jerini.de;imgt.cines.fr;及Kabatet al., Sequences of Proteins of Immunological Interest,U.S.Dept.Health(1983);各者全部內容併入本文以資參考。Examples of human architecture sequences for human adaptation are disclosed, for example, at www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.ncbi.nih.gov/igblast; www.atcc.org/phage/hdb .html;www.mrc-cpe.cam.ac.uk/ALIGNMENTS.php;www.kabatdatabase.com/top.html;ftp.ncbi.nih.gov/repository/kabat;www.sciquest.com;www.abcam .com; www.antibodyresource.com/onlinecomp.html; www.public.iastate.edu/~pedro/research_tools.html; www.whfreeman.com/immunology/CH05/kuby05.htm; www.hhmi.org/grants/ Lectures/1996/vlab;www.path.cam.ac.uk/~mrc7/mikeimages.html;mcb.harvard.edu/BioLinks/Immunology.html;www.immunologylink.com;pathbox.wustl.edu/~hcenter/ Index.html;www.appliedbiosystems.com;www.nal.usda.gov/awic/pubs/antibody;www.m.ehim-u.ac.jp/~yasuhito/Elisa.html;www.biodesign.com;www .cancerresearchuk.org; www.biotech.ufl.edu; www.isac-net.org; baserv.uci.kun.nl/~jraats/links1.html; www.recab.uni-hd.de/immuno.bme. Nwu.edu;www.mrc~cpe.cam.ac.uk;www.ibt.unam.mx/vir/V_ice.html;http://www.b Ioinf.org.uk/abs;antibody.bath.ac.uk;www.unizh.ch;www.cryst.bbk.ac.uk/~ubcg07s;www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg .html;www.path.cam.ac.uk/~mrc7/humanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stat_aim.html;www.biosci.missouri.edu/smithgp/index. Html; www.jerini.de; imgt.cines.fr; and Kabat et al., Sequences of Proteins of Immunological Interest, USD ept. Health (1983); each of which is incorporated herein by reference.
缺少任何非人類序列之完全人類單株抗體可利用下述參考文獻之技術,以人類免疫球蛋白基因轉殖小鼠製備:例如,Lonberget al.,Nature 368 :856-859(1994);Fishwildet al.,Nature Biotechnology 14 :845-851(1996)及Mendezet al.,Nature Genetics 15 :146-156(1997)。亦可利用下述參考文獻之技術製備人類單株抗體並以噬菌體展示庫予以最適化:例如,Knappiket al.,J.Mol.Biol.296 :57-86(2000)及Krebset al.,J.Immunol.Meth.254 :67-84(2001)。A fully human monoclonal antibody lacking any non-human sequence can be prepared by human immunoglobulin gene transgenic mice using the techniques of the following references: for example, Lonberg et al., Nature 368 : 856-859 (1994); Fishwild Et al., Nature Biotechnology 14 : 845-851 (1996) and Mendez et al., Nature Genetics 15 : 146-156 (1997). Human monoclonal antibodies can also be prepared using the techniques of the following references and optimized using phage display libraries: for example, Knappik et al., J. Mol. Biol. 296: 57-86 (2000) and Krebs et al. J. Immunol . Meth . 254: 67-84 (2001).
本文所用之「增殖率」一詞係指每單位時間細胞數之變化或每單位時間透過朝向細胞分裂之細胞週期展現進展標記之細胞數變化。此等標記可為形態上、DNA複製指標或經表現之基因產物。本文所用之「TLR3生物活性」或「TLR3受體活化作用」等詞係指配位體與細胞表面TLR3結果出現之任何活性。As used herein, the term "proliferation rate" refers to a change in the number of cells per unit time or a change in the number of cells exhibiting a progression marker per unit time through the cell cycle toward cell division. Such markers can be morphological, DNA replication indicators or expressed gene products. As used herein, the terms "TLR3 biological activity" or "TLR3 receptor activation" refer to any activity of the ligand and cell surface TLR3 results.
本文使用習知之一及三字母胺基酸代碼如下:
本發明乃有關能抑制由TLR3受體傳介的傳訊作用之拮抗劑及此等拮抗劑之用途。此等TLR3拮抗劑具有結合TLR3受體及抑制由TLR3受體傳介的傳訊作用等性質。可被該等拮抗劑抑制之TLR3傳訊作用之機制實例包括抑制激酶活性、轉錄減少或受體拮抗作用。能抑制利用其他機制之由TLR3受體傳介之傳訊作用之其他拮抗劑亦隸屬本發明多個態樣及具體實例範圍之內。彼等拮抗劑可作為研究試劑、診斷試劑及治療劑用。The present invention relates to antagonists which inhibit the signaling effect of TLR3 receptors and the use of such antagonists. These TLR3 antagonists have properties such as binding to the TLR3 receptor and inhibition of signaling by the TLR3 receptor. Examples of mechanisms by which TLR3 signaling can be inhibited by such antagonists include inhibition of kinase activity, transcriptional reduction, or receptor antagonism. Other antagonists that inhibit the signaling by the TLR3 receptor using other mechanisms are also within the scope of various aspects and specific examples of the invention. These antagonists can be used as research reagents, diagnostic reagents, and therapeutic agents.
本發明之一態樣為抑制選自包括IL-6、IL-8與MIP1-α的細胞介素之細胞生產之類鐸受體3(TLR3)拮抗劑。再者,本發明之拮抗劑可抑制RANTES之細胞生產。One aspect of the invention is the inhibition of a steroid receptor 3 (TLR3) antagonist produced from a cell selected from the group consisting of IL-6, IL-8 and MIP1-α. Furthermore, the antagonists of the invention inhibit the production of cells of RANTES.
於另一態樣中,本發明提供與TLR3具反應性之單離抗體,其具有單株抗體之抗原結合能力,具有如SEQ ID NOs:9(VH CDR1)、11(VH CDR2)與13(VH CDR3)所示之重鏈互補決定區(CDRs)之胺基酸序列及如SEQ ID NOs:19(VL CDR1)、21(VL CDR2)與23(VL CDR3)所示之輕鏈CDRs之胺基酸序列。一例示抗體為含有如SEQ ID NOs:9、11與13所示之重鏈CDR胺基酸序列及如SEQ ID NOs:19、21與23所示之輕鏈CDR胺基酸序列之單株抗體。In another aspect, the invention provides an isolated antibody reactive with TLR3 having the antigen binding ability of a monoclonal antibody having SEQ ID NOs: 9 ( VH CDR1), 11 ( VH CDR2) and 13 amino acid sequence (V H CDR3) of the heavy chain complementarity determining regions (CDRs) of and as shown in SEQ ID NOs: 19 (V L CDR1), 21 (V L CDR2) and 23 (V L CDR3) shown in FIG. The amino acid sequence of the light chain CDRs. An exemplary antibody is a monoclonal antibody comprising a heavy chain CDR amino acid sequence as set forth in SEQ ID NOs: 9, 11 and 13 and a light chain CDR amino acid sequence as set forth in SEQ ID NOs: 19, 21 and 23. .
本發明之另一態樣為與TLR3具反應性之單離抗體,其包含具有如SEQ ID NO:6所示胺基酸序列之VH 及具有如SEQ ID NO:16所示胺基酸序列之VL 。Another aspect of the invention is an isolated antibody reactive with TLR3 comprising a VH having the amino acid sequence set forth in SEQ ID NO: 6 and having the amino acid sequence set forth in SEQ ID NO: V L .
本發明之另一態樣為編碼本發明任何抗體或其他蛋白質TLR3拮抗劑或其補體之單離多核苷酸。本文揭示特定之多核苷酸實例,然而,於既定表現系統中具有基因密碼簡併或密碼子偏愛,編碼本發明抗體或其他蛋白質TLR3拮抗劑之其他多核苷酸亦隸屬本發明範圍之內。Another aspect of the invention is an isolated polynucleotide encoding any of the antibodies or other protein TLR3 antagonists of the invention or complement thereof. Specific polynucleotide examples are disclosed herein, however, other polynucleotides encoding an antibody of the invention or other protein TLR3 antagonists are also within the scope of the invention, with degenerate or codon bias in the established expression system.
本發明之另一態樣為含有如SEQ ID NOs:9、11與13所示CDR胺基酸序列之抗體重鏈。Another aspect of the invention is an antibody heavy chain comprising a CDR amino acid sequence as set forth in SEQ ID NOs: 9, 11 and 13.
本發明之另一態樣為編碼含有如SEQ ID NOs:19、21與23所示CDR胺基酸序列之抗體輕鏈之單離多核苷酸。Another aspect of the invention is an isolated polynucleotide encoding an antibody light chain comprising a CDR amino acid sequence as set forth in SEQ ID NOs: 19, 21 and 23.
本發明之另一態樣為編碼含有如SEQ ID NO:6所示胺基酸序列之抗體重鏈之單離多核苷酸。一多核苷酸序列實例示於SEQ ID NO:5。Another aspect of the invention is an isolated polynucleotide encoding an antibody heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 6. An example of a polynucleotide sequence is shown in SEQ ID NO:5.
本發明之另一態樣為編碼含有如SEQ ID NO:16所示胺基酸序列之抗體輕鏈之單離多核苷酸。一多核苷酸序列實例示於SEQ ID NO:15。Another aspect of the invention is an isolated polynucleotide encoding an antibody light chain comprising an amino acid sequence as set forth in SEQ ID NO: 16. An example of a polynucleotide sequence is shown in SEQ ID NO: 15.
本發明另一具體實例係適應人類之單株抗體,其含有如SEQ ID NO:25、27、29或31所示之VH 胺基酸序列及如SEQ ID NO:33、35、37或39所示之VL 胺基酸序列。編碼SEQ ID NO:25、27、29與31所示VH 胺基酸序列及SEQ ID NO:33、35、37與39所示VL 胺基酸序列之單離多核苷酸亦為本發明之態樣。彼等適應人類之單株抗體含有SEQ ID NOs:9、11與13所示之VH CDR胺基酸序列及SEQ ID NOs:19、21與23所示之VL CDR胺基酸序列。編碼SEQ ID NO:25、27、29與31之VH 胺基酸序列之核酸序列分別例示於SEQ ID NOs:26、28、30與32。編碼SEQ ID NO:33、35、37與39之VL 胺基酸序列之核酸序列分別例示於SEQ ID NOs:34、36、38與40。本發明適應人類單株抗體之一特定具體實例含有如SEQ ID NO:25所示之VH 胺基酸序列及如SEQ ID NO:33所示之VL 胺基酸序列。Another embodiment of the invention is a monoclonal antibody adapted to humans comprising a VH amino acid sequence as set forth in SEQ ID NO: 25, 27, 29 or 31 and as SEQ ID NO: 33, 35, 37 or 39 the V L amino acid sequence shown in FIG. Encoding SEQ ID NO: 25,27,29 and 31 V H amino acid sequence shown in and SEQ ID NO: 33,35,37 and single V L amino acid sequences shown in Figure 39 is also a polynucleotide of the present invention from The situation. Their adaptation of the human monoclonal antibody comprising SEQ ID NOs: 9,11 and V H CDR of the amino acid sequence shown in FIG. 13 and SEQ ID NOs: 19, 21, and 23 as shown in the amino acid sequence of V L CDR. The nucleic acid sequences encoding the VH amino acid sequences of SEQ ID NOS: 25, 27, 29 and 31 are exemplified in SEQ ID NOs: 26, 28, 30 and 32, respectively. Encoding SEQ ID NO: the amino acid sequence of a nucleic acid sequence L 33,35,37 and 39 of Example V are shown in SEQ ID NOs: 34,36,38 and 40. Specific examples of the present invention is adapted to a specific one as a human monoclonal antibody comprising SEQ ID NO: 25 H V of the amino acid sequence shown as and SEQ ID NO: The V L amino acid sequence shown in Figure 33.
本發明另一具體實例係單離抗體,其具有如下式(I)所示之VH CDR1胺基酸序列:Thr Thr Tyr Trp Xaa1 His(I)式中Xaa1 為Ile或Met(SEQ ID NO:61);如式(II)所示之VH CDR2胺基酸序列:Glu Ile Asn Pro Asn Asn Gly Arg Ile Asn Xaa2 Xaa3 Glu Lys Xaa4 Lys Thr(II)式中Xaa2 為Tyr或Gly,Xaa3 為Asn或Ala及Xaa4 為Phe或Gly(SEQ ID NO:62);及、如式(III)所示之VH CDR3胺基酸序列:Val Gly Val Xaa5 Ile Thr Thr Phe Pro Tyr(III)式中Xaa5 為Met或Ile(SEQ ID NO:63);及具有如SEQ ID NOs:19、21與23所示之胺基酸序列之VL CDRs。Another embodiment of the present invention is an isolated antibody having the VH CDR1 amino acid sequence represented by the following formula (I): Thr Thr Tyr Trp Xaa 1 His (I) wherein Xaa 1 is Ile or Met (SEQ ID) NO: 61); V H CDR2 amino acid sequence as shown in formula (II): Glu Ile Asn Pro Asn Asn Gly Arg Ile Asn Xaa 2 Xaa 3 Glu Lys Xaa 4 Lys Thr (II) where Xaa 2 is Tyr Or Gly, Xaa 3 is Asn or Ala and Xaa 4 is Phe or Gly (SEQ ID NO: 62); and, as shown in formula (III), V H CDR3 amino acid sequence: Val Gly Val Xaa 5 Ile Thr Thr Xa 5 of the formula Phe Pro Tyr (III) is Met or Ile (SEQ ID NO: 63); and VL CDRs having amino acid sequences as shown in SEQ ID NOs: 19, 21 and 23.
例示種類包括一種抗體,其具有如SEQ ID NO:33所示之VL 胺基酸序列及包含具式(I)(其中Xaa1 為Met)之VL -CDR1和分別如SEQ ID NOs:11與13所示之VL -CDR2與VL -CDR3胺基酸序列之VH 胺基酸序列(SEQ ID NO:45,例示核酸示於SEQ ID NO:46)。於此種類中,Xaa1 為Met;Xaa2 為Tyr;Xaa3 為Asn;Xaa4 為Phe;及Xaa5 為Met。Exemplary species include an antibody having a VL amino acid sequence as set forth in SEQ ID NO: 33 and comprising VL- CDR1 having the formula (I) wherein Xaa 1 is Met and SEQ ID NOs: 11 respectively and V L -CDR2 13 is shown with the V L -CDR3 amino acid sequences V H amino acid sequence (SEQ ID NO: 45, the nucleic acid is shown in exemplified SEQ ID NO: 46). In this class, Xaa 1 is Met; Xaa 2 is Tyr; Xaa 3 is Asn; Xaa 4 is Phe; and Xaa 5 is Met.
其他例示種類包括抗體,其具有如SEQ ID NO:33所示之VL 胺基酸序列和包含分別如SEQ ID NOs:9與13所示之VH -CDR1與VH -CDR3胺基酸序列及式(II)之VH -CDR2,其中:Xaa2 為Gly,Xaa3 為Asn及Xaa4 為Phe(SEQ ID NO:47,例示核酸序列示於SED ID NO:48);Xaa2 為Tyr,Xaa3 為Ala及Xaa4 為Phe(SEQ ID NO:49,例示核酸序列示於SED ID NO:50);及Xaa2 為Tyr,Xaa3 為Asn及Xaa4 為Gly(SEQ ID NO:51,例示核酸序列示於SED ID NO:52)。Other exemplary classes include antibodies having a VL amino acid sequence as set forth in SEQ ID NO: 33 and comprising VH- CDR1 and VH- CDR3 amino acid sequences as set forth in SEQ ID NOs: 9 and 13, respectively. And VH- CDR2 of the formula (II), wherein: Xaa 2 is Gly, Xaa 3 is Asn and Xaa 4 is Phe (SEQ ID NO: 47, the exemplified nucleic acid sequence is shown in SED ID NO: 48); Xaa 2 is Tyr Xaa 3 is Ala and Xaa 4 is Phe (SEQ ID NO: 49, the exemplified nucleic acid sequence is shown in SED ID NO: 50); and Xaa 2 is Tyr, Xaa 3 is Asn and Xaa 4 is Gly (SEQ ID NO: 51) The exemplified nucleic acid sequence is shown in SED ID NO: 52).
其他例示種類包括抗體,其具有如SEQ ID NO:33所示之VL 胺基酸序列和包含分別如SEQ ID NOs:9與11所示之VH -CDR1與VH -CDR2胺基酸序列及式(III)之VH -CDR3,其中Xaa5 為Ile(SEQ ID NO:53,例示核酸序列示於SEQ ID NO:54)。Other exemplary classes include antibodies having a VL amino acid sequence as set forth in SEQ ID NO: 33 and comprising VH- CDR1 and VH- CDR2 amino acid sequences as set forth in SEQ ID NOs: 9 and 11, respectively. And VH- CDR3 of formula (III), wherein Xaa 5 is Ile (SEQ ID NO: 53, an exemplary nucleic acid sequence is shown in SEQ ID NO: 54).
簡言之,例示種類包括具有下述VL
與VH
胺基酸序列組合之諸抗體:
本發明進一步包括單離抗體,其中VH 具有示於SEQ ID NO:45、47、49、51或53之胺基酸序列及VL 具有示於SEQ ID NO:33、35、37或39之胺基酸序列。The present invention further includes isolated antibodies, which has a V H are shown in SEQ ID NO: 45,47,49,51, or 53 amino acid sequence of the V L and have shown in SEQ ID NO: 33,35,37 or 39 of Amino acid sequence.
例示抗體拮抗劑可為抗體IgG、IgD、IgGA或IgM等同功型之抗體。此外,此等拮抗劑抗體可利用例如糖基化、異構化、脫糖基化,或非天然發生之共價修飾作用例如添加聚乙二醇基團(聚乙二醇化)與脂質化等方法,進行轉譯後修飾。彼等修飾作用可於活體內或試管內發生。例如,可將本發明抗體接合於聚乙二醇(PEG基化),以增進其藥物動力學性質。接合作用可利用熟習此項技藝人士已知之技術進行。已證實治療用抗體與PEG之接合增進藥物動力學性質而不干預其功能。參閱,Deckertet al.,Int.J.Cancer 87 :382-390,2000;Knightet al.,Platelets 15 :409-418,2004;Leonget al.,Cytokine 16 :106-119,2001;及Yanget al.,Protein Eng.16 :761-770,2003。Exemplary antibody antagonists can be antibodies of antibody IgG, IgD, IgGA or IgM equivalent work. Furthermore, such antagonist antibodies may utilize, for example, glycosylation, isomerization, deglycosylation, or non-naturally occurring covalent modifications such as the addition of polyethylene glycol groups (PEGylation) and lipidation. Method, post-translational modification. These modifications can occur in vivo or in vitro. For example, an antibody of the invention can be conjugated to polyethylene glycol (PEGylated) to enhance its pharmacokinetic properties. Engagement can be carried out using techniques known to those skilled in the art. The binding of therapeutic antibodies to PEG has been shown to enhance pharmacokinetic properties without interfering with its function. See, Deckert et al., Int. J. Cancer 87 : 382-390, 2000; Knight et al., Platelets 15 : 409-418, 2004; Leong et al., Cytokine 16 : 106-119, 2001; Et al., Protein Eng. 16 : 761-770, 2003.
本發明抗體之藥物動力學性質亦可透過利用熟習此項技藝人士已知技術之Fc修飾作用予以增進。舉例而言,IgG4同功型重鏈於其鉸合區域含有能形成重鏈間或重鏈內二硫鍵結之Cys-Pro-Ser-Cys(CPSC)主結構,亦即,該CPSC主結構中之二Cys殘基可與其他重鏈之對應Cys殘基形成二硫鍵結(重鏈間)或一既定CPSC主結構內之二Cys殘基可互相形成二硫鍵結(重鏈內)。一般相信,活體內異構酶能將IgG4分子之重鏈間鍵轉化為重鏈內鍵結,反之亦然(Aalberse and Schuurman,Immunology 105 :9-19(2002))。於是,由於在鉸合區域具有重鏈內鍵結之彼等IgG4分子中之重:輕鏈(HL)對彼此並非共價結合,可能解離成為HL單體,然後與衍生自其他IgG4分子之HL單體形成雙專一性、雜二聚體IgG4分子。雙專一性IgG抗體中,兩個Fabs結合之功能部位不同。以Pro取代IgG4鉸合區域中之Ser228,產生「似IgG1之作用」,亦即,該等分子於重鏈間形成穩定之二硫鍵結,因此不易與其他IgG4分子發生HL交換。於一具體實例中,本發明之諸抗體包含具有S228P突變之IgG4 Fc功能部位。The pharmacokinetic properties of the antibodies of the invention can also be enhanced by the use of Fc modifications known to those skilled in the art. For example, an IgG4 isoform heavy chain contains a Cys-Pro-Ser-Cys (CPSC) main structure capable of forming a heavy chain or a heavy intrachain disulfide bond in its hinge region, that is, the CPSC main structure Two of the Cys residues may form disulfide bonds with the corresponding Cys residues of other heavy chains (between the heavy chains) or two Cys residues within a given CPSC main structure may form a disulfide bond with each other (within the heavy chain) . It is generally believed that in vivo isomerases can convert heavy chain linkages of IgG4 molecules into heavy chain linkages and vice versa (Aalberse and Schuurman, Immunology 105 : 9-19 (2002)). Thus, due to the heavy weight of the IgG4 molecules in the hinge region having the heavy chain linkage: the light chain (HL) is not covalently bound to each other, it may dissociate into an HL monomer, and then with HL derived from other IgG4 molecules. The monomer forms a bispecific, heterodimeric IgG4 molecule. In the bispecific IgG antibody, the functional sites of the two Fabs are different. Substitution of Ser228 in the IgG4 hinge region with Pro produces "like IgG1 action", that is, the molecules form stable disulfide bonds between the heavy chains, and thus are less susceptible to HL exchange with other IgG4 molecules. In one embodiment, the antibodies of the invention comprise an IgG4 Fc functional site having a S228P mutation.
再者,可將本發明抗體中影響與除了FcRn補救受體外之Fc受體結合之位點移除。舉例而言,可將本發明抗體中涉及ADCC活性之Fc受體移除。例如,IgG1鉸合區域中之Leu234/Leu235突變成為L234A/L235A,或IgG4鉸合區域中之Phe234/Leu235突變成為P234A/L235A使FcR結合減至最小及降低免疫球蛋白傳介依賴補體的細胞毒性與ADCC之能力。於一具體實例中,本發明之諸抗體包含具有P234A/L235A突變之IgG4 Fc功能部位。Furthermore, sites in the antibodies of the invention that affect binding to Fc receptors other than the FcRn salvage receptor can be removed. For example, an Fc receptor involved in ADCC activity in an antibody of the invention can be removed. For example, a Leu234/Leu235 mutation in the IgG1 hinge region becomes L234A/L235A, or a Phe234/Leu235 mutation in the IgG4 hinge region becomes P234A/L235A to minimize FcR binding and reduce immunoglobulin-mediated complement-dependent cytotoxicity. The ability with ADCC. In one embodiment, the antibodies of the invention comprise an IgG4 Fc functional site having a P234A/L235A mutation.
於本發明之另一具體實例中,諸抗體包含具有S108P、P114A與L115A等突變之IgG4 Fc功能部位,該Fc功能部位具有示於SEQ ID NO:41之胺基酸序列。編碼SEQ ID NO:41之例示核酸序列示於SEQ ID NO:42。於全長IgG4重鏈中,同等重要之突變為S228P、P234A與L235A。In another embodiment of the invention, the antibodies comprise an IgG4 Fc functional site having a mutation such as S108P, P114A and L115A, the Fc functional site having the amino acid sequence set forth in SEQ ID NO:41. An exemplary nucleic acid sequence encoding SEQ ID NO: 41 is set forth in SEQ ID NO:42. Among the full-length IgG4 heavy chains, equally important mutations were S228P, P234A and L235A.
完全人類、適應人類、人類化及親和性成熟之抗體分子或抗體片段為隸屬本發明範圍內之模擬體、融合蛋白及嵌合蛋白。Antibody molecules or antibody fragments that are fully human, human, human, and affinity mature are mimics, fusion proteins, and chimeric proteins within the scope of the invention.
本發明拮抗劑可以小於或等於約10- 7 、10- 8 、10- 9 、10- 1 0 、10- 1 1 或10- 1 2 M之Kd 與TLR3結合。一既定分子(例如hTLR3)對於TLR3受體之親和力,可使用任何適當方法實驗測定。該等方法可利用Biacore或KinExA儀器設備、ELISA或熟習此項技藝者已知之競爭結合分析試驗。Antagonists of the invention may be less than or equal to about 10--7, 10--8, 10--9, 10--10, 10--11, or 10 - 1 2 M K d of binding to TLR3. The affinity of a given molecule (e.g., hTLR3) for the TLR3 receptor can be determined experimentally using any suitable method. Such methods can utilize Biacore or KinExA instrumentation, ELISA or competitive binding assays known to those skilled in the art.
以所需親和力結合拮抗劑分子之既定TLR3同質物可利用包括抗體親和性成熟法等技術及適用於非抗體分子之其他技藝認可之技術予以選定。A given TLR3 homologue that binds the antagonist molecule with the desired affinity can be selected using techniques including antibody affinity maturation methods and other techniques recognized for non-antibody molecules.
本發明之另一具體實例為含有本發明至少一種多核苷酸之載體。此等載體可為質體載體、病毒載體、轉因子系載體或適用於利用任何方法將本發明多核苷酸引入指定生物或基因背景內之任何其他載體。Another embodiment of the invention is a vector comprising at least one polynucleotide of the invention. Such vectors can be plastid vectors, viral vectors, transgenic vector vectors or any other vector suitable for introducing a polynucleotide of the invention into a given biological or genetic context using any method.
本發明之另一具體實例為包含本發明任何多核苷酸(例如編碼含有SEQ ID NO:9、SEQ ID NO:11與SEQ ID NO:13的多肽之多核苷酸及編碼含有SEQ ID NO:19、SEQ ID NO:21與SEQ ID NO:23的多肽之多核苷酸)之宿主細胞。其他例示宿主細胞包含編碼含有SEQ ID NOs:25、27、29、31、45、47、49、51或53之一多肽之多核苷酸及編碼含有SEQ ID NO:33、35、37或39之一多肽之多核苷酸。此等宿主細胞可為真核生物細胞、細菌細胞、植物細胞或古生細胞。例示真核生物細胞可為哺乳類、昆蟲、鳥類或其他動物來源者。哺乳類真核生物細胞包括胚質永存細胞株例如融合瘤或骨髓細胞瘤細胞株例如SP2/0(美國菌種保存中心(ATCC),Manassas,VA,CRL-1581)、NS0(European Collection of Cell Cultures(ECACC),Salisbury,Wiltshire,UK,ECACC No.85110503)、FO(ATCC CRL-1646)與Ag653(ATCC CRL-1580)鼠類細胞株。例示人類骨髓細胞瘤細胞株為U266(ATTC CRL-TIB-196)。其他有用之細胞株包括衍生自中國倉鼠卵巢(CHO)細胞例如CHO-K1(ATCC CRL-61)或DG44者。Another embodiment of the invention is a polynucleotide comprising a polynucleotide of the invention (e.g., a polynucleotide encoding a polypeptide comprising SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13 and encoding comprising SEQ ID NO: 19 Host cells of the polynucleotide of SEQ ID NO: 21 and the polypeptide of SEQ ID NO: 23. Other exemplary host cells comprise a polynucleotide encoding a polypeptide comprising one of SEQ ID NOs: 25, 27, 29, 31, 45, 47, 49, 51 or 53 and the encoding comprises SEQ ID NO: 33, 35, 37 or 39 A polynucleotide of one of the polypeptides. Such host cells may be eukaryotic cells, bacterial cells, plant cells or ancient cells. It is exemplified that eukaryotic cells can be mammals, insects, birds or other animal sources. Mammalian eukaryotic cells include germplasm perpetuating cell lines such as fusion tumors or myeloid cell lines such as SP2/0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures). (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines. The human myeloid cell line is exemplified as U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells such as CHO-K1 (ATCC CRL-61) or DG44.
本發明之另一具體實例為製造與TLR3具反應性之抗體之方法,該方法包括培養本發明之宿主細胞及回收該宿主細胞生產之抗體。此等抗體可為下文例示之為含有分別如SEQ ID NOs:6與16所示之重與輕胺基酸序列之單株抗體1068,或含有如SEQ ID NOs:25、27、29、31、45、47、49、51或53所示之重鏈胺基酸序列及如SEQ ID NOs:33、35、37或39所示之輕鏈胺基酸序列之單株抗體1068之適應人類或適應人類之CDR變異體之TLR3拮抗劑抗體。Another embodiment of the present invention is a method of producing an antibody reactive with TLR3, which comprises culturing a host cell of the present invention and recovering an antibody produced by the host cell. Such antibodies may be exemplified below as monoclonal antibodies 1068 containing heavy and light amino acid sequences as shown in SEQ ID NOs: 6 and 16, respectively, or containing SEQ ID NOs: 25, 27, 29, 31, Human or adapted to a heavy chain amino acid sequence of 45, 47, 49, 51 or 53 and a monoclonal antibody 1068 of the light chain amino acid sequence as set forth in SEQ ID NOs: 33, 35, 37 or 39 A TLR3 antagonist antibody to a human CDR variant.
本發明之另一具體實例為產生本發明抗體之融合瘤細胞株。Another embodiment of the invention is a fusion tumor cell line producing an antibody of the invention.
本發明提供預防及治療需要減少TLR3活性之症狀之方法。可使用TLR3拮抗劑治療或預防之症狀包括由細胞介素所傳介者及完全或部分由TLR3之活化或經由TLR3途徑之傳訊造成者。本發明包括抑制IL-6、IL-8或MIP1-α的細胞生產之方法,該方法包括使TLR3拮抗劑(例如本文揭示之單離抗體)與表現TLR3受體之細胞接觸足以抑制IL-6、IL-8或MIP1-α生產之時間。The present invention provides methods for preventing and treating the need to reduce the symptoms of TLR3 activity. Symptoms that can be treated or prevented using TLR3 antagonists include those caused by interleukins and those that are fully or partially activated by activation of TLR3 or via the TLR3 pathway. The invention includes a method of inhibiting cell production of IL-6, IL-8 or MIP1-α, the method comprising contacting a TLR3 antagonist (such as an isolated antibody disclosed herein) with a cell expressing a TLR3 receptor sufficient to inhibit IL-6 , IL-8 or MIP1-α production time.
本發明方法可用以治療屬於任何類別之動物病患。此等動物之實例包括哺乳動物例如人類、齧齒類、狗、貓及農場動物與其他動物種類例如鳥類、爬蟲類及魚類。於不希望拘泥於任何特定理論下,一般相信TLR3拮抗劑之治療優勢係由於此等拮抗劑抑制涉及一些炎性疾病的促炎性化學激素與細胞介素分泌之能力;亦相信TLR3拮抗劑之治療優勢係由於此等拮抗劑增加細胞增殖因而促進組織恢復之能力。The methods of the invention can be used to treat animal patients belonging to any class. Examples of such animals include mammals such as humans, rodents, dogs, cats, and farm animals and other animal species such as birds, reptiles, and fish. Without wishing to be bound by any particular theory, it is generally believed that the therapeutic advantage of TLR3 antagonists is due to the ability of these antagonists to inhibit the secretion of pro-inflammatory chemical hormones and interleukins involved in some inflammatory diseases; The therapeutic advantage is due to the ability of these antagonists to increase cell proliferation and thereby promote tissue recovery.
舉例而言,本發明方法係用於治療或預防病患之炎性疾病及促進組織恢復(例如外傷性傷害後之傷口或灼傷復原)。再者,本發明方法亦提供試管內細胞密度。For example, the methods of the invention are useful for treating or preventing an inflammatory disease in a patient and promoting tissue recovery (eg, wound or burn recovery after traumatic injury). Furthermore, the method of the invention also provides in vitro cell density.
任何TLR3拮抗劑均可於本發明之預防及治療方法中使用。舉例而言,本文揭示之任何單離抗體均可於炎性疾病之治療或預防或促進組織恢復中作為TLR3拮抗劑用。特別有用者為與TLR3具反應性之單離抗體,其具有單株抗體之抗原結合能力,含有如SEQ ID NO:9、SEQ ID NO:11與SEQ ID NO:13所示之VH CDR胺基酸序列及如SEQ ID NO:19、SEQ ID NO:21與SEQ ID NO:23所示之VL CDR胺基酸序列。其他有用之抗體包含具有如SEQ ID NOs:25、27、29、31、45、47、49、51或53所示胺基酸序列之VH 及具有如SEQ ID NOs:33、35、37或39所示胺基酸序列之VL 。Any TLR3 antagonist can be used in the prophylactic and therapeutic methods of the present invention. For example, any of the isolated antibodies disclosed herein can be used as a TLR3 antagonist in the treatment or prevention of inflammatory diseases or in promoting tissue recovery. Particularly useful are the isolated antibodies reactive with TLR3, which have the antigen binding ability of the monoclonal antibodies, and contain the VH CDR amines as shown in SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: a base acid sequence and a VL CDR amino acid sequence as set forth in SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23. Other useful antibodies comprise a VH having an amino acid sequence as set forth in SEQ ID NOs: 25, 27, 29, 31, 45, 47, 49, 51 or 53 and having SEQ ID NOs: 33, 35, 37 or V L amino acid sequences shown in Figure 39.
足以治療或預防既定炎性疾病之既定TLR3拮抗劑之量可容易地予以決定。本發明方法中,TLR3拮抗劑可單獨或與至少一種其他分子組合投與。此等附加分子可為其他TLR3拮抗劑分子或具有非由TLR3受體傳訊作用傳介之治療優勢之分子。抗生素、抗病毒劑、減少細胞介素含量或活性之緩和劑及其他化合物為該等附加分子之實例。The amount of a given TLR3 antagonist sufficient to treat or prevent a given inflammatory disease can be readily determined. In the methods of the invention, the TLR3 antagonist can be administered alone or in combination with at least one other molecule. Such additional molecules may be other TLR3 antagonist molecules or molecules having therapeutic advantages that are not mediated by TLR3 receptor signaling. Antibiotics, antiviral agents, emollients and other compounds that reduce interleukin content or activity are examples of such additional molecules.
於治療或預防炎性疾病方法之另一具體實例中,係利用抑制TLR3基因表現降低TLR3活性。TLR3基因表現可利用降低TLR3生物活性表現以抑制TLR3傳介的傳訊作用之任何方法予以抑制。此等方法包括,例如,透過與失活的基因體DNAs(例如,基因剔除、啟動子攔劫或其他基因突變方法)重組之基因不活化作用及基因轉錄不活化作用(例如,緘默RNAs或反義RNAs)。熟習此項技藝者將識別降低活性TLR3表現之許多其他方法。In another embodiment of the method of treating or preventing an inflammatory disease, the TLR3 gene activity is decreased by inhibiting the TLR3 gene expression. TLR3 gene expression can be inhibited by any method that reduces the TLR3 biological activity profile to inhibit the signaling effect of TLR3 transmission. Such methods include, for example, gene inactivation and gene transcription inactivation by recombination with inactivated genomic DNAs (eg, gene knockout, promoter occlusion, or other genetic mutation methods) (eg, silent RNAs or antisense) RNAs). Those skilled in the art will recognize many other methods of reducing the activity of active TLR3.
因此,本發明之一態樣為治療或預防炎性疾病之方法,該方法包括投與有其需要的病患治療有效量之TLR3拮抗劑足以治療或預防該炎性疾病之時間。Accordingly, one aspect of the invention is a method of treating or preventing an inflammatory disease, the method comprising administering to a patient in need thereof a therapeutically effective amount of a TLR3 antagonist sufficient to treat or prevent the inflammatory disease.
此等炎性疾病之一實例為與敗血症相關之疾病。敗血症係對感染之全身性反應,嚴重時引致器官衰竭及死亡。於醫學上,敗血症界定為由病毒、細菌、真菌、或寄生物感染產生之全身性炎性反應症候群(SIRS)。由病毒、細菌、真菌、或寄生物感染及由壞死細胞釋放之dsRNA促成敗血症之發作。與敗血症相關之疾病包括SIRS、敗血性休克或多器官功能不良症候群(MODS)。於不希望拘泥於任何特定理論下,一般相信使用TLR3拮抗劑治療,藉由延長罹患與敗血症相關的炎性疾病病患之生還時間,或防止局部炎性情況(例如,於肺中)擴散成為全身性疾病,藉由使固有抗微生物活性成為可能,藉由顯示與抗微生物劑組合使用時之增效活性,藉由使促成病變之局部炎性狀態減至最小程度,或前述任何組合,可提供治療優勢。此等介入足以容許確保病患生存需要之附加治療(例如治療潛藏之感染或減少細胞介素含量)。An example of such an inflammatory disease is a disease associated with sepsis. Septicemia is a systemic response to infection that causes organ failure and death in severe cases. In medicine, sepsis is defined as systemic inflammatory response syndrome (SIRS) produced by infection with viruses, bacteria, fungi, or parasites. Infection with viruses, bacteria, fungi, or parasites and dsRNA released by necrotic cells contribute to the onset of sepsis. Diseases associated with sepsis include SIRS, septic shock, or multiple organ dysfunction syndrome (MODS). Without wishing to be bound by any particular theory, it is generally believed that treatment with TLR3 antagonists may result in prolonged survival of patients with inflammatory diseases associated with sepsis or prevention of local inflammatory conditions (eg, in the lungs). Systemic diseases, by making intrinsic antimicrobial activity possible, by exhibiting synergistic activity when used in combination with an antimicrobial agent, by minimizing the local inflammatory state contributing to the lesion, or any combination of the foregoing Provide therapeutic advantages. Such interventions are sufficient to allow for additional treatment (eg, treatment of latent infections or reduced interleukin levels) that warrants the patient's survival.
此等炎性疾病之另一實例為炎性腸道疾病,此炎性腸道疾病可為克隆氏症或潰瘍性結腸炎。熟習此項技藝者可識別已知或未知病因之引起腸道發炎之其他炎性腸道疾病。再者,TLR3拮抗劑可用於治療及預防與潰瘍性結腸炎或克隆氏症相關之腸道外後遺症例如關節痛與關節炎,包括僵直性脊椎炎、薦腸關節炎與牛皮癬性脊椎關節炎。其他腸道外後遺症包括黏膜傷害例如口腔潰瘍、結節性紅斑(形成疼痛硬化卵形瘤)與特徵為皮膚極嚴重潰瘍之壞疽性膿皮症;眼睛併發症例如鞏膜炎、虹膜炎與葡萄膜炎;腎臟疾病例如腎結石;肝膽疾病例如原發性硬化膽管炎、與潰瘍性結腸炎或克隆氏症相關之特徵為纖維變性發炎之慢性肝臟疾病;及為長期使用皮質類固醇發生的併發症之骨骼疾病包括骨質疏鬆症與骨質缺乏症;亦包括由IBD誘發之肺部功能不良及呼吸疾病包括間質性肺炎、氣管狹窄、細支氣管炎、細支氣管阻塞性組織化肺炎、肺部血管炎、類肉瘤病、慢性支氣管炎、及具有嗜曙紅血球過多之肺部浸潤之臨床疾病。Another example of such inflammatory diseases is an inflammatory bowel disease, which may be Crohn's disease or ulcerative colitis. Those skilled in the art will be able to identify other inflammatory bowel diseases that cause intestinal inflammation, either known or unknown. Furthermore, TLR3 antagonists are useful in the treatment and prevention of parenteral sequelae associated with ulcerative colitis or Crohn's disease such as arthralgia and arthritis, including ankylosing spondylitis, enteric arthritis and psoriatic spondyloarthritis. Other extraintestinal sequelae include mucosal injuries such as oral ulcers, nodular erythema (formation of pain-sclerosing ovarian tumors) and gangrenous pyoderma characterized by extremely severe skin ulcers; ocular complications such as scleritis, iritis and uveitis; Kidney diseases such as kidney stones; hepatobiliary diseases such as primary sclerosing cholangitis, chronic liver diseases characterized by fibrotic inflammation associated with ulcerative colitis or Crohn's disease; and bone diseases that are complications of long-term use of corticosteroids Includes osteoporosis and osteopenia; also includes pulmonary dysfunction and respiratory diseases induced by IBD including interstitial pneumonia, tracheal stenosis, bronchiolitis, bronchiole obstructive tissue pneumonitis, pulmonary vasculitis, sarcoma Disease, chronic bronchitis, and clinical disease with pulmonary infiltration of eosinophilic red blood cells.
此等炎性疾病之另一實例為與感染相關之疾病,其包含病毒性或細菌性肺炎,包括嚴重肺炎、囊胞性纖維症、支氣管炎、氣道惡化與急性呼吸窘迫症候群(ARDS)。該等與感染相關之疾病尚包括重複感染例如原發性病毒感染及續發性細菌感染。Another example of such inflammatory diseases is infection-related diseases, including viral or bacterial pneumonia, including severe pneumonia, cystic fibrosis, bronchitis, airway deterioration, and acute respiratory distress syndrome (ARDS). Such infection-related diseases also include repeated infections such as primary viral infections and secondary bacterial infections.
此等炎性疾病之另一實例為炎性肺部疾病,其實例包含由感染誘發之肺部疾病,包括與病毒、細菌、真菌、寄生物或傳染性蛋白質等感染相關者;由過敏原誘發的肺部疾病;由污染物誘發的肺部疾病例如石棉沉著症、矽肺症、或鈹肺症;由胃抽吸誘發的肺部疾病;免疫失調;由基因誘發的炎性肺部疾病例如囊胞性纖維症;及由身體受傷誘發的肺部疾病,例如呼吸器傷害。彼等炎性疾病亦包含氣喘、肺氣腫、支氣管炎、COPD、類肉瘤病、組織細胞增生症、淋巴管肌瘤增生、急性肺傷害、急性呼吸窘迫症候群、慢性肺疾、支氣管肺部異常、社區感染之肺炎、院內感染之肺炎、呼吸器相關肺炎、敗血症、病毒性肺炎、流行性感冒感染、副流感感染、人類間質肺病毒感染、呼吸道融合病毒感染及麴黴菌或其他真菌感染。Another example of such inflammatory diseases is inflammatory lung disease, examples of which include infection-induced lung diseases, including those associated with viruses, bacteria, fungi, parasites or infectious proteins; induced by allergens Pulmonary diseases; pulmonary diseases induced by pollutants such as asbestosis, silicosis, or silicosis; pulmonary diseases induced by gastric aspiration; immune disorders; inflammatory lung diseases induced by genes such as pouches Cytoplasmic fibrosis; and lung disease caused by physical injury, such as respiratory injury. These inflammatory diseases also include asthma, emphysema, bronchitis, COPD, sarcoma, histiocytosis, lymphangioma, acute lung injury, acute respiratory distress syndrome, chronic lung disease, bronchopulmonary abnormalities , community-infected pneumonia, nosocomial pneumonia, respirators related pneumonia, sepsis, viral pneumonia, influenza infection, parainfluenza infection, human interstitial lung virus infection, respiratory syncytial virus infection, and fungal or other fungal infections.
此等炎性疾病之另外實例為2型糖尿病、血脂失調(dislipidemia)及代謝症候群。TLR3拮抗劑係用於抑制與肥胖及胰島素抗阻相關之炎性過程。抑制TLR3傳訊作用會改善病患之血脂狀況,亦即降低總膽固醇含量及增加HDlc/LDLc比率。抑制TLR3傳訊作用亦將導致胰島素分泌增加因而導致改善胰島素抗阻。目前之2型糖尿病治療與多種有害副作用相關,包括血糖過低及體重增加。使用TLR3拮抗劑治療2型糖尿病被預期具有較少的副作用及持久之藥物動力性質。再者,以具有長循環半衰期之化合物(例如本發明單離抗體)進行治療,不需要經常用藥,有助於改善用藥順從性-為2型糖尿病之重大議題。Additional examples of such inflammatory diseases are type 2 diabetes, dislipidemia, and metabolic syndrome. TLR3 antagonists are used to inhibit inflammatory processes associated with obesity and insulin resistance. Inhibition of TLR3 signaling will improve the patient's blood lipid profile, which is to reduce total cholesterol and increase HDlc/LDLc ratio. Inhibition of TLR3 signaling will also result in increased insulin secretion leading to improved insulin resistance. Current treatments for type 2 diabetes are associated with a variety of harmful side effects, including hypoglycemia and weight gain. Treatment of type 2 diabetes with a TLR3 antagonist is expected to have fewer side effects and sustained pharmacokinetic properties. Furthermore, treatment with compounds having a long circulating half-life (e.g., isolated antibodies of the invention) does not require frequent administration and contributes to improved medication compliance - a major issue for type 2 diabetes.
同時,改善血脂狀況有延緩或防止與肥胖及2型糖尿病相關的心血管疾病(例如動脈硬化)進展的可能。此外,抑制TLR3傳訊作用經由直接影響胰臟小島細胞或者藉由影響血脂狀況及保護小島細胞免受高脂含量引致之惡化,可能增加胰島素之流通量。因此,單獨抑制TLR3或組合其他療法有於2型糖尿病中延緩引入胰島素治療及避開與胰島素治療相關之不為所欲之副作用的可能。At the same time, improving blood lipid status may delay or prevent the progression of cardiovascular diseases (such as arteriosclerosis) associated with obesity and type 2 diabetes. In addition, inhibition of TLR3 signaling may increase insulin flux by directly affecting pancreatic islet cells or by affecting blood lipid status and protecting islet cells from high fat content. Thus, inhibition of TLR3 alone or in combination with other therapies may delay the introduction of insulin therapy and avoid the undesirable side effects associated with insulin therapy in type 2 diabetes.
再者,罹患C型肝炎與HIV感染的病患由於在肝臟中累積脂質,容易進展為胰島素抗阻及2型糖尿病。利用TLR3拮抗劑抑制TLR3傳訊作用可於此高度危及之病患族群中同時攻擊該感染及胰島素抗阻。Furthermore, patients with hepatitis C and HIV infection are prone to progress to insulin resistance and type 2 diabetes due to accumulation of lipids in the liver. Inhibition of TLR3 signaling by TLR3 antagonists can simultaneously challenge the infection and insulin resistance in this highly endangered patient population.
可利用本發明方法預防或治療之其他炎性疾病與神經病變包括多發性硬化症、硬化症、紅斑性狼瘡、及神經退化性與中樞神經系(CNS)失調症包括老年癡呆症、帕金森氏症、亨丁頓氏舞蹈症(Huntington’s disease)、躁鬱症與肌萎縮性脊髓側索硬化症(ALS)、肝臟疾病包括纖維變性、肝炎C病毒(HCV)與肝炎B病毒(HBV)、關節炎、、類風濕性關節炎、牛皮癬性關節炎與幼年型類風濕性關節炎(JRA)、骨質疏鬆症、骨關節炎、胰臟炎、纖維變性、腦炎、牛皮癬、巨細胞性動脈炎、僵直性脊椎炎、自體免疫性肝炎、人類免疫缺乏病毒(HIV)、炎性皮膚疾病、移植、癌症、過敏症、內分泌疾病、其他自體免疫失調症及氣道過度反應。Other inflammatory diseases and neuropathies that may be prevented or treated by the methods of the invention include multiple sclerosis, sclerosis, lupus erythematosus, and neurodegenerative and central nervous system (CNS) disorders including Alzheimer's disease, Parkinson's disease Disease, Huntington's disease, bipolar disorder and amyotrophic lateral sclerosis (ALS), liver disease including fibrosis, hepatitis C virus (HCV) and hepatitis B virus (HBV), arthritis , rheumatoid arthritis, psoriatic arthritis and juvenile rheumatoid arthritis (JRA), osteoporosis, osteoarthritis, pancreatitis, fibrosis, encephalitis, psoriasis, giant cell arteritis, Ankylosing spondylitis, autoimmune hepatitis, human immunodeficiency virus (HIV), inflammatory skin diseases, transplantation, cancer, allergies, endocrine diseases, other autoimmune disorders, and airway hyperreactivity.
本發明之其他態樣為增加細胞增殖率之方法,該方法包括利用,例如使細胞與TLR3拮抗劑接觸,降低細胞中之TLR3活性。於本發明此態樣之一具體實例中,細胞可為得自例如上皮或結腸組織之細胞。上皮細胞可源自任何上皮組織,舉例而言,例如,消化道上皮、皮膚上皮、肺上皮、或支氣管肺部上皮。炎性疾病可能影響任何組織,舉例而言,例如,心臟組織及消化道組織,導致結構上與功能上偏離正常組織。於一些情況下,此等炎性疾病可能係遺傳因素或感染的結果。於其他情形下,此等炎性疾病可能係外傷性傷害(舉例而言,例如,灼傷)的結果。熟習此項技藝者將認知許多不同的炎性疾病及相關病變由涉及之不同組織展現。A further aspect of the invention is a method of increasing the rate of cell proliferation comprising, for example, contacting a cell with a TLR3 antagonist to reduce TLR3 activity in the cell. In one embodiment of this aspect of the invention, the cells can be cells derived from, for example, epithelial or colonic tissue. Epithelial cells can be derived from any epithelial tissue, for example, the digestive tract epithelium, the cutaneous epithelium, the lung epithelium, or the bronchopulmonary epithelium. Inflammatory diseases can affect any tissue, for example, heart tissue and digestive tract tissue, resulting in structural and functional deviations from normal tissue. In some cases, such inflammatory diseases may be the result of genetic factors or infections. In other instances, such inflammatory diseases may be the result of traumatic injuries, for example, burns. Those skilled in the art will recognize that many different inflammatory diseases and related disorders are exhibited by different tissues involved.
本發明之另一態樣為治療起因於細胞死亡的疾病之方法,該方法包括投與有其需要的病患治療有效量之TLR3拮抗劑足以治療該疾病之時間。Another aspect of the invention is a method of treating a condition resulting from cell death, the method comprising administering to a patient in need thereof a therapeutically effective amount of a TLR3 antagonist sufficient to treat the condition.
本發明之另一態樣為預防起因於細胞死亡的疾病之方法,該方法包括投與有其需要的病患治療有效量之TLR3拮抗劑足以預防該疾病之時間。Another aspect of the invention is a method of preventing a disease caused by cell death comprising administering to a patient in need thereof a therapeutically effective amount of a TLR3 antagonist sufficient to prevent the disease.
供治療用途之本發明拮抗劑之投與方式可為遞送製劑至宿主之任何適當途徑。蛋白質、抗體、抗體片段與模擬體及彼等製劑之醫藥組成物特別可供非經腸投與用,亦即,經皮下、肌內、皮膚內、靜脈內或經鼻內遞送。The dosage of the antagonist of the invention for therapeutic use can be any suitable route for delivery of the formulation to the host. The pharmaceutical compositions of proteins, antibodies, antibody fragments and mimetics and their formulations are especially useful for parenteral administration, i.e., subcutaneous, intramuscular, intradermal, intravenous or intranasal delivery.
本發明之拮抗劑可於醫藥上可接受之載劑中製備為含有有效量拮抗劑作為活性成分之醫藥組成物。以呈備用注射形式之含拮抗劑之水性懸浮液或溶液為佳,較佳為於生理pH之緩衝液。供非經腸投與之組成物通常包含溶於醫藥上可接受之載劑(較佳為水性載劑)中之本發明拮抗劑之溶液或其混合物。可使用多種水性載劑,例如,0.4%食鹽水、0.3%甘胺酸等。彼等溶液為無菌,通常不含微粒物質。彼等溶液可利用習用、悉知之殺菌技術(例如,過濾法)殺菌。組成物可依需要含有醫藥上可接受之輔助物質,例如pH調整劑及緩衝劑等,俾使接近生理狀況。於此等醫藥組成物中,本發明拮抗劑之濃度可大為不同,亦即,可小於約0.5%,通常為或至少約1%至多達15或20重量%,主要可基於流體容積、黏性等,根據所選擇之特定投與方式予以選定。The antagonist of the present invention can be prepared as a pharmaceutical composition containing an effective amount of an antagonist as an active ingredient in a pharmaceutically acceptable carrier. Preferably, the aqueous suspension or solution containing the antagonist is in the form of a ready injection, preferably a buffer at physiological pH. Compositions for parenteral administration typically comprise a solution of an antagonist of the invention dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier, or a mixture thereof. A variety of aqueous carriers can be used, for example, 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and usually free of particulate matter. These solutions can be sterilized using conventional, well-known sterilization techniques (eg, filtration). The composition may contain pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers as needed to achieve a near physiological condition. In such pharmaceutical compositions, the concentrations of the antagonists of the present invention can vary widely, that is, can be less than about 0.5%, typically at least or at least about 1% up to 15 or 20% by weight, based primarily on fluid volume, viscosity. Sex, etc., selected according to the specific mode of investment chosen.
因此,供肌內注射用之本發明醫藥組成物可製備為含有1毫升無菌緩衝水,及介於約1奈克至約100毫克,例如,約50奈克至約30毫克或更佳為約5毫克至約25毫克之本發明拮抗劑。同樣地,供注入靜脈內用之本發明醫藥組成物可製成含有約250毫升無菌林格氏溶液,及約1毫克至約30毫克及更佳為約5毫克至約25毫克之本發明拮抗劑。製備非經腸投與用組成物之實際方法為一般悉知及更具細節地見述於,例如,”Remington’s Pharmaceutical Sciences”,15th ed.,Mack Publishing Company,Easton,PA。Accordingly, the pharmaceutical composition of the present invention for intramuscular injection can be prepared to contain 1 ml of sterile buffered water, and preferably from about 1 ng to about 100 mg, for example, from about 50 ng to about 30 mg or more. 5 mg to about 25 mg of an antagonist of the invention. Similarly, the pharmaceutical compositions of the present invention for intravenous administration can be formulated to contain about 250 milliliters of sterile Ringer's solution, and from about 1 milligram to about 30 milligrams, and more preferably from about 5 milligrams to about 25 milligrams of the antagonist of the present invention. Agent. The actual methods of preparing compositions for parenteral administration are generally known and described in more detail, for example, "Remington's Pharmaceutical Sciences", 15th ed., Mack Publishing Company, Easton, PA.
於醫藥製劑中,本發明之拮抗劑可呈單位劑量型存在。適當之治療有效劑量可容易地由熟習此項技藝人士決定。如果需要,則決定之劑量可於治療期間,依醫師選定之適當時間間隔重複投與。In pharmaceutical preparations, the antagonists of the invention may be presented in unit dosage form. Suitable therapeutically effective doses can be readily determined by those skilled in the art. If desired, the determined dose can be administered repeatedly at appropriate intervals selected by the physician during treatment.
本發明之拮抗劑可凍乾貯存,使用之前,於適當載劑中再組成。此技術已被證實對於習知免疫球蛋白類及蛋白質製劑有效,及可使用此項技藝中已知之凍乾及再組成技術。The antagonists of the invention can be stored lyophilized and reconstituted in a suitable carrier prior to use. This technique has proven to be effective for conventional immunoglobulins and protein preparations, and freeze-drying and reconstitution techniques known in the art can be used.
拮抗劑可利用提供此等分子至細胞之任何技術投與。就細胞而言,舉例而言,可藉由在培養基中補充該拮抗劑而進行試管內拮抗劑投與;藉由將靜脈內注射該拮抗劑至動物或組織中進行活體內拮抗劑投與。熟習此項技藝者將認知試管內或活體內投與拮抗劑至細胞之其他方式。此等方式亦包括上文詳述之遞送製劑至宿主之彼等方式。Antagonists can be administered using any technique that provides such molecules to the cell. In the case of cells, for example, in-line antagonist administration can be carried out by supplementing the antagonist in a medium; in vivo antagonist administration by intravenous injection of the antagonist into an animal or tissue. Those skilled in the art will recognize other ways of administering an antagonist to a cell in vitro or in vivo. These methods also include the manner in which the formulations are delivered to the host as detailed above.
茲參照下文之具體、非限制實例敘述本發明。The invention is described with reference to specific, non-limiting examples that follow.
抗 -hTLR3 拮抗劑單株抗體之鑑定 利用細胞系篩檢分析法鑑定能封阻經由hTLR3受體之傳訊作用之抗-hTLR3拮抗劑單株抗體。使用標準技術(Kohleret al., 1976),於BALB/C小鼠中產生製造抗-hTLR3單株抗體之一群融合瘤。利用經皮膚內注射編碼hTLR3胺基酸1-703之質體DNA(SEQ ID NO:3),對小鼠進行hTLR3之免疫處理。胺基酸1-703(SEQ ID NO:4)相當於預測之hTLR3胞外功能部位。先使用10微克質體DNA對小鼠進行注射,兩週後,進行第二次之10微克DNA注射。此第二次10微克質體DNA注射兩週後,投與各小鼠15微克DNA之追加注射。於B細胞融合三天前,以於磷酸鹽緩衝鹽液(PBS;10 mM磷酸鹽、150 mM NaCl,pH 7.4)中之15微克hTLR3蛋白質,對小鼠進行靜脈內注射。收取經免疫處理的小鼠之脾臟,使用標準方法(Kohleret al., 1976),進行B細胞融合。使用含次黃嘌呤-胺基喋呤-胸腺核苷之培養基選擇融合瘤,先利用酵素結合免疫吸附分析法(ELISA)篩檢抗-TLR3。利用限制稀釋法選殖製造抗-hTLR3單株抗體之個別融合瘤。 Identification of anti- hTLR3 antagonist monoclonal antibodies Cell line screening assays were used to identify anti- hTLR3 antagonist monoclonal antibodies that block the signaling via the hTLR3 receptor. A fusion of one of the anti-hTLR3 monoclonal antibodies was produced in BALB/C mice using standard techniques (Kohler et al., 1976). Mice were immunized with hTLR3 by intradermal injection of plastid DNA encoding hTLR3 amino acid 1-703 (SEQ ID NO: 3). Amino acid 1-703 (SEQ ID NO: 4) corresponds to the predicted extracellular functional site of hTLR3. Mice were first injected with 10 micrograms of plastid DNA, and two weeks later, a second 10 micrograms of DNA injection was performed. Two weeks after the second 10 micrograms of plastid DNA injection, an additional injection of 15 micrograms of DNA from each mouse was administered. Three days prior to B cell fusion, mice were injected intravenously with 15 micrograms of hTLR3 protein in phosphate buffered saline (PBS; 10 mM phosphate, 150 mM NaCl, pH 7.4). The spleens of the immunized mice were harvested and subjected to B cell fusion using standard methods (Kohler et al., 1976). The fusion tumor was selected using a medium containing hypoxanthine-aminopurine-thymidine, and the anti-TLR3 was first screened by enzyme-binding immunosorbent assay (ELISA). Individual fusion tumors of anti-hTLR3 monoclonal antibodies were produced by restriction dilution.
藉由使用人類A549衍生之肺上皮細胞株穩定大量表現之hTLR3之細胞系篩檢分析法,鑑定製造抗-TLR3拮抗劑單株抗體之融合瘤。A fusion cell producing an anti-TLR3 antagonist monoclonal antibody was identified by using a cell line screening assay for hTLR3 in which a large number of human lung A549-derived lung epithelial cell lines were stably expressed.
用以產生供彼等分析法用之篩檢及對照細胞株之A549細胞(ATCC CRL:CCL-185)係得自美國菌種保存中心(Manassas,VA)。篩檢用細胞株係命名A549-hTLR3之A549衍生細胞株。以編碼hTLR3與抗新黴素基因之哺乳類質體表現載體穩定轉染A549-hTLR3細胞。對照組A549衍生細胞株命名A549-neo。單僅以編碼該抗新黴素基因之哺乳類質體表現載體穩定轉染A549-neo細胞。根據廠商之操作指南及標準篩檢與選殖方法,利用Lipofectamine(Invitrogen,Inc.,Carlsbad,CA)轉染,產生彼等穩定轉染之細胞株。標準條件下,於含10% FBS、1% MEM非必要胺基酸(Gibco Invitrogen,Inc.,Carlsbad,CA)、1 mM麩胺醯胺、1 mM丙酮酸鈉、20 mM HEPES與0.5毫克/毫升G418之最低營養要求培養基(MEM)中培養A549-hTRL3及A549-neo細胞。A549 cells (ATCC CRL: CCL-185) used to generate screening and control cell lines for their assays were obtained from the American Type Culture Collection (Manassas, VA). The cell line for screening was named A549-hTLR3 A549-derived cell line. A549-hTLR3 cells were stably transfected with a mammalian plastid expression vector encoding hTLR3 and anti-neomycin gene. The control A549-derived cell line was named A549-neo. A549-neo cells were stably transfected only with a mammalian plastid expression vector encoding the anti-neomycin gene. Use Lipofectamine according to the manufacturer's operating guidelines and standard screening and selection methods (Invitrogen, Inc., Carlsbad, CA) transfected to produce their stably transfected cell lines. Under standard conditions, containing 10% FBS, 1% MEM non-essential amino acid (Gibco Invitrogen, Inc., Carlsbad, CA), 1 mM glutamine, 1 mM sodium pyruvate, 20 mM HEPES and 0.5 mg / A549-hTRL3 and A549-neo cells were cultured in milliliters of G418 minimum nutrient requirement medium (MEM).
使用A549-hTLR3細胞進行細胞系篩檢分析法,鑑定出一hTLR3拮抗劑單株抗體,命名mAb 1068。構成彼等篩檢分析法之基礎原則為多(I:C)刺激存在A549-hTLR3細胞中之hTLR3受體,導致細胞介素生產增加。相較於未暴露於單株抗體之對照組A549-hTLR3細胞,經由篩檢分析法鑑定出之候選hTLR3拮抗劑單株抗體可於A549-hTLR3細胞中透過hTLR3受體抑制由多(I:C)傳介之傳訊作用,及引致降低之細胞介素生產。A549-hTLR3 cells were used for cell line screening assay to identify a hTLR3 antagonist monoclonal antibody, named mAb 1068. The underlying principle that constitutes their screening assay is that multiple (I:C) stimulation of the hTLR3 receptor in A549-hTLR3 cells results in increased interleukin production. Compared to the control group A549-hTLR3 cells that were not exposed to monoclonal antibodies, the candidate hTLR3 antagonist monoclonal antibodies identified by screening assay could be inhibited by the hTLR3 receptor in A549-hTLR3 cells (I:C). ) the role of messaging and the resulting reduction in interleukin production.
篩檢分析法之進行係在添加5微克/毫升多(I:C)(Amersham Biosciences Corp.,Piscataway,NJ)之前,於37℃培育具有測試單株抗體之A549-hTLR3細胞30分鐘;24小時後,測定細胞培養物上澄液中之細胞介素量。同法處理對照組A549-hTLR3細胞,惟彼等細胞不與測試單株抗體一起培育。根據廠商指導,使用Luminex多通道分析(Luminex Corp.,Austin,TX)及接合IL-6(介白素-6)、IL-8(介白素-8)、與RANTES(根據活化作用調控,正常地T-表現,及據推測分泌之)專一單株抗體之珠粒測定篩檢分析法中之細胞介素產量。利用此等分析法鑑定hTLR3與拮抗劑mAb 1068之結合作用。The screening assay was performed by incubating A549-hTLR3 cells with the test monoclonal antibody for 30 minutes at 37 °C before adding 5 μg/ml (I:C) (Amersham Biosciences Corp., Piscataway, NJ); 24 hours; Thereafter, the amount of interleukin in the cell culture supernatant was determined. The control group A549-hTLR3 cells were treated in the same manner, but the cells were not incubated with the test monoclonal antibodies. Use Luminex according to manufacturer's instructions Multichannel analysis (Luminex Corp., Austin, TX) and conjugation of IL-6 (interleukin-6), IL-8 (interleukin-8), and RANTES (regulated by activation, normal T-expression, And the interleukin production in the bead assay screening assay for the specific monoclonal antibody that is presumed to be secreted. These assays were used to identify the binding of hTLR3 to the antagonist mAb 1068.
使用標準方法,自表現mAb 1068之融合瘤選殖編碼mAb 1068之重鏈與輕鏈之重鏈與輕鏈核酸序列。mAb 1068重鏈與輕鏈核酸及胺基酸序列分別示於圖1與2及SEQ ID NOs:6與16。使用標準方法,產生包含編碼重組mAb 1068(r1068)之重鏈與輕鏈核酸序列之細胞株。The heavy and light chain nucleic acid sequences encoding the heavy and light chains of mAb 1068 were cloned from a fusion tumor expressing mAb 1068 using standard methods. The mAb 1068 heavy and light chain nucleic acid and amino acid sequences are shown in Figures 1 and 2 and SEQ ID NOs: 6 and 16, respectively. Cell lines containing the heavy and light chain nucleic acid sequences encoding recombinant mAb 1068 (r1068) were generated using standard methods.
hTLR3 拮抗劑於人類肺衍生細胞中抑制 IL-6 、 IL-8 與 RANTES 等細胞介素生產之作用 hTLR3拮抗劑mAb 1068於人類肺上皮衍生之A549-hTLR3細胞中抑制由hTLR3-傳介之IL-6(圖3)、IL-8(圖4)與PANTES(圖5)等細胞介素之生產。然而,於彼等人類肺衍生細胞中,hTLR3專一鼠類mAb TLR3.7(eBioscience,San Diego,CA)不抑制hTLR3傳介、多(I:C)誘發之IL-6(圖3)、IL-8(圖4)與RANTES(圖5)之生產。由於先前之工作暗示TLR3.7單株抗體可能拮抗hTLR3受體(Matsurnoto M.et al.,Biochem.Biophys Res.Commun.24 :1364-1369(2002)),因此1068與TLR3.7單株抗體間之此項區別很重要。此先前工作記述TLR3.7單株抗體似乎在人類纖維母細胞衍生MRC-5細胞中抑制多(I:C)誘發的IFN-β之生產(Matsumoto M.et al.,Biochem.Biophys Res.Commun.24 :1364-1369(2002))。本文之結果清楚地指出,1068 hTLR3拮抗劑單株抗體比TLR3.7單株抗體抑制更寬廣的細胞介素譜之生產,且可據此將此二單株抗體互相區分。 hTLR3 antagonist inhibits IL-6 , IL-8 and RANTES and other interleukin production in human lung-derived cells hTLR3 antagonist mAb 1068 inhibits IL- mediated by hTLR3- in human lung epithelial-derived A549-hTLR3 cells Production of interleukins such as -6 (Fig. 3), IL-8 (Fig. 4) and PANTES (Fig. 5). However, in their human lung-derived cells, the hTLR3-specific murine mAb TLR3.7 (eBioscience, San Diego, CA) did not inhibit hTLR3-mediated, multi- (I:C)-induced IL-6 (Fig. 3), IL. Production of -8 (Fig. 4) and RANTES (Fig. 5). Since previous work suggested that TLR3.7 monoclonal antibodies may antagonize the hTLR3 receptor (Matsurnoto M. et al., Biochem. Biophys Res. Commun. 24 : 1364-1369 (2002)), 1068 and TLR3.7 monoclonal antibodies This distinction between the two is important. This previous work describes that TLR3.7 monoclonal antibodies appear to inhibit multi (I:C)-induced IFN-β production in human fibroblast-derived MRC-5 cells (Matsumoto M. et al., Biochem. Biophys Res. Commun .24 : 1364-1369 (2002)). The results herein clearly indicate that the 1068 hTLR3 antagonist monoclonal antibody inhibits the production of a broader interleukin profile than the TLR3.7 monoclonal antibody, and the two monoclonal antibodies can be distinguished from one another.
IL-6、IL-8與RANTES專一細胞介素分析法之進行係如圖3、圖4與圖5所示,在添加5微克/毫升多(I:C)(Amersham Biosciences Corp.,Piscataway,NJ)之前,於37℃培育具有1068單株抗體或TLR3.7單株抗體之A549-hTLR3細胞30分鐘。24小時後,酌情使用Luminex儀器設備(Luminex Corp.,Austin,TX)及接合IL-6、IL-8或RANTES專一單株抗體之珠粒測定細胞培養物上澄液中之細胞介素量。各細胞介素之Luminex分析法係根據廠商指示進行。The IL-6, IL-8 and RANTES-specific intercellular assays were performed as shown in Figures 3, 4 and 5, with the addition of 5 μg/ml (I:C) (Amersham Biosciences Corp., Piscataway, Prior to NJ), A549-hTLR3 cells with 1068 monoclonal antibodies or TLR3.7 monoclonal antibodies were incubated at 37 °C for 30 minutes. Use Luminex as appropriate after 24 hours The amount of interleukin in the cell culture supernatant was determined by instrumentation (Luminex Corp., Austin, TX) and beads conjugated to IL-6, IL-8 or RANTES specific monoclonal antibodies. Luminex of each interleukin The analysis method is based on the manufacturer's instructions.
hTLR3 拮抗劑於初級人類支氣管-上皮細胞中抑制 MIP1-α 與 IL-6 細胞介素之生產 hTLR3拮抗劑mAb 1068於初級人類支氣管-上皮細胞中抑制由hTLR3-傳介之MIP1-α(圖6)與IL-6(圖7)等細胞介素之生產。 hTLR3 antagonist inhibits MIP1-α and IL-6 interleukin production in primary human bronchial-epithelial cells hTLR3 antagonist mAb 1068 inhibits MIP1-α - mediated by hTLR3- in primary human bronchial-epithelial cells (Fig. 6 ) Production of interleukins such as IL-6 (Figure 7).
MIP1-α與IL-6專一細胞介素分析法之進行係如圖6或圖7所示,在添加5微克/毫升多(I:C)(Amersham Biosciences Corp.,Piscataway,NJ)之前,於37℃培育具有1068單株抗體或非專一性多株小鼠IgG製劑之初級人類支氣管-上皮細胞細胞30分鐘。24小時後,酌情使用Luminex儀器設備(Luminex Corp.,Austin,TX)及接合MIP1-α或IL-6專一單株抗體之珠粒測定細胞培養物上澄液中之細胞介素量。各細胞介素之Luminex分析法係根據廠商指示進行。初級人類支氣管-上皮細胞係使用標準方法,自人類組織試樣單離。The MIP1-α and IL-6-specific intercellular assays were performed as shown in Figure 6 or Figure 7, prior to the addition of 5 μg/ml (I:C) (Amersham Biosciences Corp., Piscataway, NJ). Primary human bronchial-epithelial cells with 1068 monoclonal antibodies or non-specific multiple mouse IgG preparations were incubated at 37 °C for 30 minutes. Use Luminex as appropriate after 24 hours The amount of interleukin in the cell culture supernatant was determined by instrumentation (Luminex Corp., Austin, TX) and beads conjugated with MIP1-α or IL-6 specific monoclonal antibodies. Luminex of each interleukin The analysis method is based on the manufacturer's instructions. Primary human bronchial-epithelial cell lines were isolated from human tissue samples using standard methods.
剔除 TLR3 活性減緩炎性腸道疾病症狀之嚴重性 於剔除TLR3受體基因活性(圖8)之IBD鼠類模式中,炎性腸道疾病(IBD)症狀之嚴重性降低。克隆氏症及潰瘍性結腸炎可使用已攝入硫酸鈉葡聚醣(DSS)之動物為模式(Hendrickson B.A.et al.,Clin Microbiol Rev.15 :79-94,2002)。於彼等動物模式中觀察到的症狀包括體重大量減少(圖8)與上皮細胞潰瘍。彼等症狀模擬於罹患IBD(例如潰瘍性結腸炎或克隆氏症)之病患中觀察到之彼等症狀。相較於DSS處理之野生型小鼠,於此IBD鼠類模式中,DSS處理之TLR3剔除小鼠體重未大量減少(圖8),並顯露較溫和之上皮細胞傷害(根據組織病理學分析)。彼等結果顯示,TLR3傳訊作用於炎性過程中(例如涉及IBD者)扮演決定性角色。 Elimination of TLR3 activity slows the severity of symptoms of inflammatory bowel disease The severity of inflammatory bowel disease (IBD) symptoms is reduced in the IBD murine model in which TLR3 receptor gene activity (Figure 8) is removed. Crohn's disease and ulcerative colitis can be modeled using animals that have ingested sodium sulfate dextran (DSS) (Hendrickson BA et al., Clin Microbiol Rev. 15:79-94, 2002). Symptoms observed in their animal models included a significant reduction in body weight (Figure 8) and epithelial cell ulcers. These symptoms mimic the symptoms observed in patients with IBD (eg, ulcerative colitis or Crohn's disease). Compared to DSS-treated wild-type mice, DSS-treated TLR3 knockout mice did not significantly reduce body weight in this IBD murine model (Fig. 8) and revealed milder epithelial cell damage (according to histopathological analysis) . Their results show that TLR3 signaling plays a decisive role in the inflammatory process (eg, involving IBD).
於彼等實驗中,野生型C57BL/6公小鼠或TLR3剔除小鼠(Alexopoulouet al.,Nature,413 :732-738(2001))各如圖8所示,任意給與含5%(w/v)硫酸鈉葡聚醣(DSS)之飲用水或未補充之水5天,以誘發急性潰瘍性結腸炎。所有小鼠均為6-8週大,各處理組均具有至少5隻小鼠。藉由觀察體重(圖9)、結腸重量、糞便硬度、直腸出血與結腸免疫組織病理學等變化,評估DSS處理後之結腸炎進展。所有此等評估均根據已建立之動物照料及使用指導方針進行。圖8中之數據以處理1至5天之重量變化百分比表示。各符號代表得自一隻小鼠之數據。WT標明野生型小鼠;KO標明TLR剔除小鼠。橫條代表平均值;所示數據為三個獨立實驗之合成。對照組野生型及未接受DSS之TLR3基因剔除小鼠(圖8)顯示類似之重量變化(P=0.06,t-試驗)。野生型及接受DSS之TLR3基因剔除小鼠(圖8)明顯地顯示不同之重量變化(P=0.003,t-試驗)。In these experiments, wild-type C57BL/6 male mice or TLR3 knockout mice (Alexopoulou et al., Nature, 413 : 732-738 (2001)) are shown in Figure 8, and any given dose is 5% ( w/v) Potassium sulfate dextran (DSS) in drinking water or unsupplemented water for 5 days to induce acute ulcerative colitis. All mice were 6-8 weeks old and each treatment group had at least 5 mice. The progression of colitis after DSS treatment was assessed by observing changes in body weight (Figure 9), colon weight, stool hardness, rectal bleeding, and colonic immunohistochemistry. All such assessments are based on established guidelines for animal care and use. The data in Figure 8 is expressed as a percentage change in weight for 1 to 5 days of treatment. Each symbol represents data from a mouse. WT indicated wild type mice; KO indicated TLR knockout mice. The bars represent the mean; the data shown is a synthesis of three independent experiments. TLR3 knockout mice (Fig. 8) with wild-type and no DSS in the control group showed similar weight changes (P = 0.06, t-test). Wild-type and TLR3 knockout mice receiving DSS (Fig. 8) clearly showed different weight changes (P = 0.003, t-test).
於實驗第5天,收取動物結腸供組織病理學分析用。使用標準方法,將結腸包埋於石蠟中,切片,以蘇木素與曙紅染色。得自接受DSS的野生型小鼠之代表性結腸切片展現黏膜潰瘍及稠密之炎性浸潤以及腺窩與杯狀細胞喪失。得自接受未補充之水的TLR3基因剔除小鼠之代表性結腸切片具有與接受未補充之水的野生型小鼠之結腸所觀察類似之形態與組織學。得自接受DSS的TLR3基因剔除小鼠之代表性結腸包含一些稠密之細胞浸潤,惟除此之外展現未受損傷之黏膜上皮及極微之杯狀細胞喪失。此組織病理數據指出,接受DSS之TLR3基因剔除小鼠較接受DSS之野生型小鼠形成較少之上皮潰瘍,顯示TLR3活性可於炎性過程中(例如涉及IBD者)扮演決定性角色。On day 5 of the experiment, the animal colon was harvested for histopathological analysis. The colon was embedded in paraffin, sectioned, and stained with hematoxylin and eosin using standard methods. Representative colon sections from wild-type mice receiving DSS exhibited mucosal ulcers and dense inflammatory infiltrates as well as loss of glandular and goblet cells. Representative colon sections from TLR3 knockout mice receiving unsupplemented water have similar morphology and histology as observed in the colon of wild type mice receiving unsupplemented water. Representative colonies from TLR3 knockout mice receiving DSS contained some dense cell infiltration, but in addition showed uninjured mucosal epithelium and minimal goblet cell loss. This histopathological data indicated that TLR3 knockout mice receiving DSS formed less epithelial ulcers than wild-type mice receiving DSS, indicating that TLR3 activity may play a decisive role in inflammatory processes (eg, involving IBD).
hTLR3 拮抗劑處理終止炎性腸道疾病相關之減重 於IBD鼠類模式中,hTLR3拮抗劑處理降低炎性腸道疾病(IBD)相關的減重之嚴重性(圖9)。數據顯示,以TLR3拮抗劑處理可減弱與IBD(例如潰瘍性結腸炎及克隆氏症)相關之症狀。此外,此結果進一步指出,TLR3傳訊作用可於炎性疾病例如IBD中扮演重要角色。 hTLR3 antagonist treatment to stop weight loss associated with inflammatory bowel disease In the IBD murine model, hTLR3 antagonist treatment reduced the severity of inflammatory bowel disease (IBD)-related weight loss (Figure 9). The data show that treatment with TLR3 antagonists attenuates symptoms associated with IBD (eg, ulcerative colitis and Crohn's disease). In addition, this result further indicates that TLR3 signaling can play an important role in inflammatory diseases such as IBD.
於彼等實驗中,野生型C57BL/6公小鼠各如圖9所示,任意給與含5%(w/v)硫酸鈉葡聚醣(DSS)之飲用水或未補充之水5天,以誘發急性潰瘍性結腸炎。如圖9所示,於DSS處理最初4天,每天對小鼠進行0.2毫克mAb 1068於PBS載劑中、0.2毫克非專一性小鼠IgG多株抗體製劑於PBS載劑中、或單獨PBS載劑之腹膜內注射。各注射液由於PBS中之0.9毫升單株抗體或非專一性IgG製劑或單獨0.9毫升PBS載劑組成。所有小鼠均為6-8週大,各處理組含有至少5隻小鼠。In these experiments, wild-type C57BL/6 male mice were each given as shown in Figure 9, and given 5% (w/v) sodium sulfate dextran (DSS) in drinking water or unsupplemented water for 5 days. To induce acute ulcerative colitis. As shown in Figure 9, mice were given 0.2 mg mAb 1068 in PBS vehicle, 0.2 mg of non-specific mouse IgG polyclonal antibody preparation in PBS vehicle, or PBS alone for the first 4 days of DSS treatment. Intraperitoneal injection of the agent. Each injection consisted of 0.9 ml of monoclonal antibody or non-specific IgG preparation in PBS or 0.9 ml of PBS vehicle alone. All mice were 6-8 weeks old and each treatment group contained at least 5 mice.
藉由觀察體重(圖8)、結腸重量、糞便硬度、直腸出血、與結腸免疫組織病理學等變化,評估DSS處理後之結腸炎進展。所有此等評估均根據已建立之動物照料及使用指導方針進行。The progression of colitis after DSS treatment was assessed by observing changes in body weight (Fig. 8), colon weight, stool hardness, rectal bleeding, and colonic immunohistopathology. All such assessments are based on established guidelines for animal care and use.
圖9中之數據以處理1至4天之重量變化百分比表示。各符號代表得自一隻小鼠之數據。橫條代表平均值;所示數據為兩個獨立實驗之合成。The data in Figure 9 is expressed as a percentage change in weight for 1 to 4 days of treatment. Each symbol represents data from a mouse. The bars represent the mean; the data shown is a synthesis of two independent experiments.
接受DSS與mAb 1068之小鼠及未接受DSS之小鼠間,其重量變化無顯著差異[P<<0.05,唐氏試驗(Dunn’s test);圖9]。接受DSS與mAb 1068的小鼠之重量變化則與接受DSS與於PBS中之非專一性IgG或單獨PBS之小鼠觀察所得有顯著差異(二者均為P<0.01;唐氏試驗;圖9)。There was no significant difference in weight change between mice receiving DSS and mAb 1068 and mice not receiving DSS [P<<0.05, Dunn's test; Figure 9]. The change in weight of mice receiving DSS and mAb 1068 was significantly different from that observed in mice receiving DSS with non-specific IgG or PBS alone in PBS (both P < 0.01; Down's test; Figure 9) ).
hTLR3 拮抗劑處理小鼠中,慢性結腸炎嚴重性降低 所有研究均使用六至八週大之野生型C57BL/6母小鼠與出身背景為C57BL/6之TLR3剔除(KO)小鼠(Alexopoulouet al.,Nature 413 :732-738,(2001))。給與小鼠合計三個週期之於飲用水中之2%(wt/vol)硫酸鈉葡聚醣(DSS)(Okayasuet al.,Gastroenterology 98 :694-702(1990))。任意給與DSS水5天,以誘發急性潰瘍性結腸炎,然後給與未摻雜之飲用水(白水)9天。第14天開始2%DSS之第二個5天週期,隨後休息9天。第28天開始2%DSS之第三個週期,此次為7天。在兩個不同時間點犧牲小鼠:於進行研究第25天進入第二個週期休息後,或者於進行研究第37天第三個DSS週期之後。各處理組由至少8隻小鼠組成。藉由觀察整個研究期間之體重變化以及犧牲後之其他評估參數包括結腸長度、結腸重量、糞便硬度、直腸出血、與結腸免疫組織病理學等變化,評估DSS處理後之結腸炎進展。 Chronic colitis was reduced in mice treated with hTLR3 antagonists. All studies used wild-type C57BL/6 female mice of six to eight weeks old and TLR3 knockout (KO) mice of the background C57BL/6 (Alexopoulou et Al., Nature 413 : 732-738, (2001)). The mice were given three cycles of 2% (wt/vol) sodium sulfate dextran (DSS) in drinking water (Okayasu et al., Gastroenterology 98 :694-702 (1990)). The DSS water was given arbitrarily for 5 days to induce acute ulcerative colitis, and then undoped drinking water (white water) was given for 9 days. On the 14th day, the second 5-day cycle of 2% DSS was started, followed by a 9-day break. The third cycle of 2% DSS started on the 28th day, this time is 7 days. The mice were sacrificed at two different time points: after the second cycle of the study on the 25th day, or after the third DSS cycle on the 37th day of the study. Each treatment group consisted of at least 8 mice. The progression of colitis after DSS treatment was assessed by observing changes in body weight throughout the study period and other assessment parameters after sacrifice including colon length, colon weight, stool hardness, rectal bleeding, and colonic immunohistopathology.
由不清楚上述研究設計之獨立獸醫病理學家進行組織病理學評估。針對結腸之縱向切片進行一組變化之計分,包括上皮細胞壞死、上皮潰瘍與蛻皮、腺窩喪失、腺窩細胞增生、於固有層形成肉芽組織、黏膜下層之肉芽組織、黏膜下層炎性細胞浸潤及黏膜下層水腫。反映傷害擴大之計分如下:0,不存在;1,溫和,病灶;2,溫和,多病灶;3,中等,常發現,惟於有限區域;4,嚴重,於提交組織之許多區域常發現;5,很嚴重,擴大至提交組織之大部分。Histopathological evaluation was performed by an independent veterinary pathologist who was not aware of the above study design. Scoring a set of changes in the longitudinal section of the colon, including epithelial cell necrosis, epithelial ulcer and ecdysis, loss of glandular fossa, glandular cell hyperplasia, formation of granulation tissue in the lamina propria, granulation tissue of the submucosa, submucosal inflammatory cells Infiltration and submucosal edema. The scores reflecting the expansion of injury are as follows: 0, no presence; 1, mild, lesions; 2, mild, multi-focal; 3, medium, often found, only in limited areas; 4, severe, often found in many areas of the submitted organization ;5, very serious, expanded to the majority of the submitted organization.
使用學生氏試驗(Student’s test)(JMP,SAS Institute;GraphPad Prism)進行統計分析。罹患潰瘍性結腸炎及克隆氏症的病患之症狀包括體重減輕、出現便血、及結腸上皮層潰瘍。因此硫酸鈉葡聚醣-處理小鼠誘發之症狀部分模擬罹患潰瘍性結腸炎或克隆氏症病患所見症狀(Hendricksonet al.,Clin.Microbiol.Rev.15 :79-94(2002))。Statistical analysis was performed using Student's test (JMP, SAS Institute; GraphPad Prism). Symptoms of patients with ulcerative colitis and Crohn's disease include weight loss, blood in the stool, and colonic epithelial ulcers. Thus, the symptoms induced by sodium sulfate dextran-treated mice partially mimic symptoms seen in patients with ulcerative colitis or Crohn's disease (Hendrickson et al., Clin. Microbiol. Rev. 15 :79-94 (2002)).
於此模式中,野生型及TLR3 KO小鼠攝入DSS之各週期體重均減少。然而,TLR3 KO小鼠之減重情形比野生型小鼠不顯著。根據利用結腸發炎及傷害總測量之評估,TLR3 KO小鼠亦顯示較小之疾病嚴重性:TLR3 KO小鼠之結腸縮短比WT小鼠所見明顯較少;且TLR3 KO小鼠直腸出血頻率減少很多。結腸黏膜傷害之組織病理學評估與彼等總測量一致。就單一細胞壞死、上皮潰瘍、上皮蛻皮、腺窩脫離及腺窩膿腫之中位計分而言,TLR3 KO小鼠比WT小鼠更低。彼等數據加在一起顯示缺少TLR3傳訊作用於慢性結腸炎小鼠模式中授予部分疾病保護作用,及暗示TLR3傳訊作用有加重人類IBD嚴重性之可能。In this model, wild-type and TLR3 KO mice lost weight in each cycle of DSS intake. However, the weight loss of TLR3 KO mice was not significant compared to wild type mice. TLR3 KO mice also showed less disease severity based on assessment of total inflammation and injury using colon: TLR3 KO mice had significantly fewer colon shortenings than WT mice; and TLR3 KO mice had a much reduced rectal bleeding frequency . The histopathological assessment of colonic mucosal injury was consistent with their overall measurements. TLR3 KO mice were lower than WT mice in terms of single cell necrosis, epithelial ulcer, epithelial ecdysis, glandular detachment, and glandular abscess score. Together, these data show that the lack of TLR3 signaling confers partial disease protection in a mouse model of chronic colitis, and suggests that TLR3 signaling may increase the severity of human IBD.
為了進一步證明TLR3於疾病調節上之角色,乃以拮抗劑抗-TLR3 mAb 1068處理WT C57BL/6小鼠。預防性(於第一個DSS週期開始,「Pr」)或「治療性」(於第二個DSS週期開始,「Th」;圖35)地使接觸DSS小鼠組接受0.2毫克抗-TLR3 mAb 1068。接觸DSS小鼠之對照組接受PBS(載劑對照組)或0.2毫克非專一性負對照組單株抗體。另一組對照組未給與DSS。圖35之星號代表抗-TLR3拮抗劑單株抗體用藥之諸時間點。To further demonstrate the role of TLR3 in disease regulation, WT C57BL/6 mice were treated with the antagonist anti-TLR3 mAb 1068. Prophylactic (starting with the first DSS cycle, "Pr") or "therapeutic" (starting at the second DSS cycle, "Th"; Figure 35) to expose the DSS group to receive 0.2 mg of anti-TLR3 mAb 1068. The control group exposed to DSS mice received PBS (vehicle control group) or 0.2 mg of non-specific negative control monoclonal antibody. Another group of controls was not given DSS. The asterisk of Figure 35 represents the time points of administration of the anti-TLR3 antagonist monoclonal antibody.
所有接觸DSS小鼠組別於攝取DSS之第一週期後,減重即告減少(圖36)。圖36中各符號代表至少八隻小鼠之平均值,誤差棒代表標準偏差。於第0至4天、第14至18天及第28至35天給與DSS。然而,以抗-TLR3單株抗體處理組相較於以PBS處理組或單株抗體對照組,顯示減重情形較少,且於第二DSS週期後體重回復速率較快(圖37)。抗-TLR3單株抗體處理組於第三DSS週期後之減重情形大為減少(圖38)。接受PBS或對照組單株抗體之接觸DSS小鼠,從研究開始(第0天)至研究結束(第37天),平均淨減重大約20%。以抗-TLR3單株抗體處理明顯降低大約10%之減重(圖39)。圖39中,所示數據為從研究開始(第0天)至研究結束(第37天)之體重變化%;正數值顯示淨增重,負數值顯示淨減重。抗-TLR3單株抗體處理組之減重%明顯地小於以對照組載劑(PBS)或非專一性IgG處理之組別[預防性抗-TLR3處理(抗-TLR3 P)vs.PBS,P=0.006;抗-TLR3 P vs.非專一性IgG,P=0.006);治療性抗-TLR3(抗-TLR3 Th)vs.PBS,P=0.001;抗-TLR3 Th vs.非專一性IgG,P=0.009]。各符號代表一隻小鼠;橫條代表平均值。All exposure to DSS mice was reduced after weight loss in the first cycle of DSS (Figure 36). The symbols in Figure 36 represent the average of at least eight mice and the error bars represent the standard deviation. DSS was administered on Days 0 to 4, Days 14 to 18, and Days 28 to 35. However, the anti-TLR3 monoclonal antibody-treated group showed less weight loss than the PBS-treated group or the monoclonal antibody control group, and the body weight recovery rate was faster after the second DSS cycle (Fig. 37). The weight loss of the anti-TLR3 monoclonal antibody treatment group after the third DSS cycle was greatly reduced (Fig. 38). DSS mice receiving PBS or control monoclonal antibodies received an average net weight loss of approximately 20% from the start of the study (Day 0) to the end of the study (Day 37). Treatment with anti-TLR3 monoclonal antibody significantly reduced weight loss by approximately 10% (Figure 39). In Fig. 39, the data shown are % change in body weight from the start of the study (Day 0) to the end of the study (Day 37); positive values indicate net weight gain, and negative values indicate net weight loss. The % weight loss of the anti-TLR3 monoclonal antibody-treated group was significantly smaller than that of the control vehicle (PBS) or non-specific IgG [prophylactic anti-TLR3 treatment (anti-TLR3 P) vs. PBS, P =0.006; anti-TLR3 P vs. non-specific IgG, P=0.006); therapeutic anti-TLR3 (anti-TLR3 Th) vs. PBS, P=0.001; anti-TLR3 Th vs. non-specific IgG, P =0.009]. Each symbol represents a mouse; the bars represent the mean.
抗-TLR3單株抗體處理亦減少結腸縮短長度。預防性或者治療性地以抗-TLR3單株抗體處理的小鼠組別之結腸長度別明顯大於給與載劑或對照組單株抗體之組別(圖40)(抗-TLR3 P vs.PBS,P=0.009;抗-TLR3 P vs.非專一性IgG,P=0.01;抗-TLR3 Th vs.PBS,P=0.03;抗-TLR3 Th vs.非專一性IgG,P=0.04)。Anti-TLR3 monoclonal antibody treatment also reduced colon shortening length. The colon length of the mouse group treated prophylactically or therapeutically with anti-TLR3 monoclonal antibody was significantly greater than that of the vehicle alone or the control group (Figure 40) (anti-TLR3 P vs. PBS) , P=0.009; anti-TLR3 P vs. non-specific IgG, P=0.01; anti-TLR3 Th vs. PBS, P=0.03; anti-TLR3 Th vs. non-specific IgG, P=0.04).
再者,根據溫和組織病理學變化(包括上皮細胞壞死、腺窩脫離、上皮潰瘍與蛻皮、腺窩喪失與腺窩細胞增生)及慢性修復性組織病理學變化(包括於固有層形成肉芽組織、黏膜下層之肉芽組織、黏膜下層炎性細胞浸潤與黏膜下層水腫;圖41a)評估,相較於給與PBS或非專一性對照組單株抗體之對照組,以抗-TLR3單株抗體治療性處理之組別,其結腸黏膜傷害顯然較不嚴重。圖式中所示數據代表接受DSS及不同處理(組別:1,經PBS載劑處理;3,預防性抗-TLR3單株抗體;4,治療性抗-TLR3單株抗體;5,非專一性對照組單株抗體)的各組小鼠之所有組織病理學計分總和、溫和變化總和、或慢性變化總和。各圖式右側之圓圈涵蓋各處理組別計分之平均值及標準偏差。各組別間統計學上之顯著差異以具有最小重疊之圓圈表現。Furthermore, according to mild histopathological changes (including epithelial cell necrosis, glandular detachment, epithelial ulcer and epithelium, loss of glandular fossa and glandular cell proliferation) and chronic repair histopathological changes (including formation of granulation tissue in the lamina propria, The granulation tissue, submucosal inflammatory cell infiltration and submucosal edema in the submucosa; Figure 41a) Evaluation, anti-TLR3 monoclonal antibody therapeutic compared to the control group given PBS or non-specific control monoclonal antibody In the treated group, the colonic mucosal damage is obviously less serious. The data shown in the figure represents DSS and different treatments (group: 1, treated with PBS carrier; 3, prophylactic anti-TLR3 monoclonal antibody; 4, therapeutic anti-TLR3 monoclonal antibody; 5, non-specific The sum of all histopathological scores, the sum of mild changes, or the sum of chronic changes in each group of mice of the control group. The circle on the right side of each figure covers the mean and standard deviation of the scores for each treatment group. Statistically significant differences between groups are presented in circles with minimal overlap.
特別是,相較於PBS或非專一性單株抗體,抗-TLR3單株抗體處理減少上皮潰瘍及防止黏膜下層與固有層中肉芽組織之形成(圖41b)。圖式中所示數據代表接受DSS及不同處理(組別:1,經PBS載劑處理;3,預防性抗-TLR3單株抗體;4,治療性抗-TLR3單株抗體;5,非專一性對照組單株抗體)的各組小鼠之組織病理學計分。各圖式右側之圓圈涵蓋各處理組別計分之平均值及標準偏差。各組別間統計學上之顯著差異性以具有最小重疊之圓圈表現。In particular, anti-TLR3 monoclonal antibody treatment reduced epithelial ulceration and prevented the formation of granulation tissue in the submucosa and lamina propria compared to PBS or non-specific monoclonal antibodies (Fig. 41b). The data shown in the figure represents DSS and different treatments (group: 1, treated with PBS carrier; 3, prophylactic anti-TLR3 monoclonal antibody; 4, therapeutic anti-TLR3 monoclonal antibody; 5, non-specific Histopathological scoring of mice in each group of the control group. The circle on the right side of each figure covers the mean and standard deviation of the scores for each treatment group. Statistically significant differences between groups are represented by circles with minimal overlap.
為了確定潛在免疫與抗-TLR3-授予之保護相關,乃檢測免疫細胞族群及全身細胞介素量。觀察到接觸DSS與脾臟及腸繫膜淋巴結中活化T細胞數之增加相關(圖42),符合說明T細胞涉及此慢性結腸炎模式之已公告報告。使用流式細胞技術測量分別代表全身及局部T細胞活化作用之脾臟及腸繫膜淋巴結中CD62Ll o w T細胞之頻率。慢性結腸炎與脾臟及腸繫膜淋巴結中活化CD4+(助手)T細胞頻率增加相關,暗示助手T細胞活化作用全面增加。脾臟中活化CD8+具效應T細胞之頻率降低伴隨腸繫膜淋巴結中活化CD8+ T細胞頻率增加,暗示具效應T細胞流通至腸部位。從第25天,第二DSS週期之後,示出數據。各符號代表得自一隻小鼠之數據;橫條代表平均值。To determine the potential immunity associated with the protection conferred by the anti-TLR3-, the immune cell population and the amount of systemic interleukins were detected. Exposure to DSS was observed to be associated with an increase in the number of activated T cells in the spleen and mesenteric lymph nodes (Figure 42), consistent with an published report indicating that T cells are involved in this chronic colitis pattern. The frequency of CD62L l o w T cells in the spleen and mesenteric lymph nodes representing systemic and local T cell activation, respectively, was measured using flow cytometry. Chronic colitis is associated with increased frequency of activated CD4+ (helper) T cells in the spleen and mesenteric lymph nodes, suggesting an overall increase in the activation of helper T cells. Decreased frequency of activated CD8+ effector T cells in the spleen is accompanied by an increase in the frequency of activated CD8+ T cells in the mesenteric lymph nodes, suggesting that the effector T cells circulate to the gut. From day 25, after the second DSS cycle, the data is shown. Each symbol represents data from one mouse; the bars represent the average.
此外,於接觸DSS小鼠之脾臟中發現CD11b+細胞頻率較高,可能反映炎性巨噬細胞中結腸炎相關之增加。顯著地,預防性抗-TLR3單株抗體處理與脾臟CD11b+細胞頻率明顯減少至未接觸DSS之對照組小鼠所見含量相關(圖43)。接觸DSS之抗-TLR3單株抗體處理小鼠脾臟中之CD11b+細胞百分比與未接受DSS之小鼠類似,且顯著地低於接受PBS(P=0.001)或非專一性IgG(P=0.02)之接觸DSS小鼠。從第25天,第二DSS週期之後,示出數據。各符號代表得自一隻小鼠之數據;橫條代表平均值。Furthermore, the high frequency of CD11b+ cells found in the spleens of DSS-exposed mice may reflect an increase in colitis associated with inflammatory macrophages. Significantly, prophylactic anti-TLR3 monoclonal antibody treatment was associated with a significant reduction in spleen CD11b+ cell frequency to the levels seen in control mice not exposed to DSS (Figure 43). The percentage of CD11b+ cells in the spleens of mice treated with anti-TLR3 monoclonal antibody exposed to DSS was similar to that of mice not receiving DSS, and was significantly lower than that of PBS (P=0.001) or non-specific IgG (P=0.02). Contact with DSS mice. From day 25, after the second DSS cycle, the data is shown. Each symbol represents data from one mouse; the bars represent the average.
接觸DSS小鼠之血清細胞介素概況亦顯示與抗-TLR3單株抗體處理相關之改變:於預防性接受抗-TLR3單株抗體之小鼠中測得增加之IL-4及IL-10含量(圖44)。於誘發慢性DSS結腸炎期間之抗-TLR3單株抗體處理提高全身性IL-4及IL-10含量。所示數據分別代表第2及第3 DSS週期後之時間點,第25天及第37天,之數據。各符號代表得自一隻小鼠之數據;橫條代表平均值。IL-4與IL-10二者均被證明於炎性之調控上扮演關鍵角色。觀察到IL-10剔除小鼠自然形成結腸炎,暗示IL-10於控制IBD免疫致病機轉中之專一角色。彼等結果暗示,抗-TLR3單株抗體處理改變由DSS攝取誘發的炎性及T細胞反應。Serum interleukin profiles in exposed DSS mice also showed changes associated with anti-TLR3 monoclonal antibody treatment: increased IL-4 and IL-10 levels in mice receiving prophylactic anti-TLR3 monoclonal antibodies (Figure 44). Anti-TLR3 monoclonal antibody treatment during induction of chronic DSS colitis increased systemic IL-4 and IL-10 levels. The data shown represent the data at the time points after the 2nd and 3rd DSS cycles, on days 25 and 37, respectively. Each symbol represents data from one mouse; the bars represent the average. Both IL-4 and IL-10 have been shown to play a key role in the regulation of inflammation. It was observed that IL-10 knockout mice naturally developed colitis, suggesting that IL-10 plays a specific role in controlling the transition of the IBD immune pathogenesis machine. These results suggest that anti-TLR3 monoclonal antibody treatment alters the inflammatory and T cell responses induced by DSS uptake.
總之,彼等數據證明,以抗-TLR3單株抗體封阻TLR3傳訊作用可於慢性結腸炎模式中改善疾病嚴重性,及提供抗-TLR3單株抗體治療人類IBD之潛在效力之證據。Taken together, these data demonstrate that blocking TLR3 signaling with anti-TLR3 monoclonal antibodies may improve disease severity in chronic colitis mode and provide evidence of the potential efficacy of anti-TLR3 monoclonal antibodies in the treatment of human IBD.
hTLR3 拮抗劑處理增加敗血症存活 利用投與D-半乳糖胺及多(I:C),可使用動物(例如小鼠)作為敗血症模式。於此等模式中,D-半乳糖胺係一種肝毒素,其功能為敗血症「致敏劑」;多(I:C)為敗血症誘發分子,模擬dsRNA及活化TLR3。結果指示,於敗血症鼠類模式中,TLR3拮抗劑處理幾乎使動物存活率加倍。 Treatment with hTLR3 Antagonists Increases Septicemia Survival By administering D-galactosamine and multiple (I:C), animals (e.g., mice) can be used as a mode of sepsis. In these models, D-galactosamine is a hepatotoxin whose function is sepsis "sensitizer"; poly(I:C) is a sepsis-inducing molecule, mimics dsRNA and activates TLR3. The results indicate that TLR3 antagonist treatment almost doubled animal survival in the septic rat model.
如圖10所示,於彼等實驗中,野生型C57BL/6母小鼠給與1毫克hTLR3拮抗劑1068 mAb於PBS載劑中、1毫克非專一性鼠類多株IgG製劑於PBS載劑中、或單獨PBS載劑之腹膜內注射。各注射液由於PBS中之1毫升單株抗體或非專一性IgG製劑或單獨1毫升PBS載劑組成。如圖10所示,第二天使小鼠接受10微克多(I:C)與20毫克D-半乳糖胺(Sigma-Aldrich Corp.,St.Louis,MO)於100微升無菌PBS中之腹膜內注射。每天兩次,監測小鼠之存活3天。所有評估均根據已建立之動物照料及使用指導方針進行。結果顯示,hTLR3拮抗劑處理增加敗血症鼠類模式中之動物存活率(圖10)。As shown in Figure 10, in their experiments, wild-type C57BL/6 female mice were given 1 mg of hTLR3 antagonist 1068 mAb in PBS vehicle, 1 mg of non-specific murine IgG preparation in PBS vehicle. Intraperitoneal injection of medium or PBS alone. Each injection consisted of 1 ml of monoclonal antibody or non-specific IgG preparation in PBS or 1 ml of PBS alone. As shown in Figure 10, the second angelic mouse received 10 micrograms (I:C) and 20 milligrams of D-galactosamine (Sigma-Aldrich Corp., St. Louis, MO) in 100 microliters of sterile PBS. Injection inside. The mice were monitored for survival for 3 days twice daily. All assessments are based on established guidelines for animal care and use. The results showed that hTLR3 antagonist treatment increased the survival rate of animals in the sepsis murine model (Fig. 10).
hTLR3 拮抗劑處理於敗血症鼠類模式中降低 IL-6 及 TNF-α 細胞介素生產 於敗血症鼠類模式中,hTLR3拮抗劑處理降低與炎症相關之IL-6(圖11)及TNF-α(圖12)細胞介素之血清含量。此結果指示,抑制TLR3活性可藉由降低TLR3傳介之促成敗血症的細胞介素之生產而促進敗血症存活。 hTLR3 antagonist treatment reduced IL-6 and TNF-α interleukin production in a septic mouse model in a septic mouse model, and hTLR3 antagonist treatment reduced inflammation-associated IL-6 (Figure 11) and TNF-α (Fig. 11) Figure 12) Serum content of interleukins. This result indicates that inhibition of TLR3 activity promotes sepsis survival by reducing the production of interleukins that are promoted by TLR3.
投與多(I:C)兩小時後,利用CO2 /O2 麻醉小鼠後眼窩竇採血法,製備得自如實例6所述處理的小鼠之血清,其製備係於室溫培育血液,隨後於2500 rpm離心15分鐘。進行細胞介素分析試驗之前,於-80℃貯存血清。酌情使用Luminex儀器設備(Luminex Corp.,Austin,TX)及接合IL-6(圖11)或TNF-α(圖12)專一單株抗體之珠粒測定血清試樣中之細胞介素含量。各細胞介素之Luminex分析法係根據廠商指示進行。所有評估均根據已建立之動物照料及使用指導方針進行。Two hours after the administration of multiple (I:C), the mice were treated with the spleen sinus blood collection method by anesthetizing the CO 2 /O 2 to prepare the blood of the mouse treated as described in Example 6, and the preparation was performed by culturing blood at room temperature. It was then centrifuged at 2500 rpm for 15 minutes. Serum was stored at -80 °C prior to the interleukin assay. Use Luminex as appropriate The interleukin content in the serum samples was determined by instrumentation (Luminex Corp., Austin, TX) and beads conjugated to IL-6 (Figure 11) or TNF-α (Figure 12) specific monoclonal antibodies. Luminex of each interleukin The analysis method is based on the manufacturer's instructions. All assessments are based on established guidelines for animal care and use.
圖11與圖12中之各符號代表得自一隻小鼠之數據。橫條代表平均值;所示數據為兩個獨立實驗之合成。投與多(I:C)兩小時後,以mAb 1068處理顯著減少血清IL-6含量(P=0.04,t-試驗;圖11);投與多(I:C)兩小時後,以mAb 1068處理顯著減少血清TNF-α含量(P=0.03,t-試驗;圖12)。The symbols in Figures 11 and 12 represent data from a mouse. The bars represent the mean; the data shown is a synthesis of two independent experiments. Two hours after administration of multiple (I:C), treatment with mAb 1068 significantly reduced serum IL-6 levels (P=0.04, t-test; Figure 11); administration of multiple (I:C) two hours later, with mAb Treatment with 1068 significantly reduced serum TNF-[alpha] levels (P = 0.03, t-test; Figure 12).
多 I:C 投與誘發促炎性細胞介素之分泌及向上調控肺中之 TLR 基因表現 每24小時,使經異佛蘭(Isoflurane)麻醉處理之野生型公或母C57BL/6小鼠接受多(I:C)於PBS中之三種劑量或單獨PBS之鼻內投與三天。所有小鼠均為12週大。如表1所示,各多(I:C)劑量含有50微克或100微克多(I:C);各劑量容積為50微升。各處理組別含有6至8隻小鼠。利用CO2 處理犧牲小鼠,於最後一次劑量24小時後,進行肺插管。於肺中注入1毫升PBS,進行支氣管沖洗(BAL),回收流出物。然後離心彼等BAL製劑使細胞沉澱,收集不含細胞之上澄液,貯存於-80℃至供多通道細胞介素分析試驗用。所有評估均根據已建立之動物照料及使用指導方針進行。 Multiple I:C administration induces secretion of pro-inflammatory interleukins and up-regulates TLR gene expression in the lungs every 24 hours, allowing wild-type male or female C57BL/6 mice treated with isoflurane anesthesia Multiple (I:C) were administered intranasally in three doses in PBS or PBS alone for three days. All mice were 12 weeks old. As shown in Table 1, each (I:C) dose contained 50 micrograms or more than 100 micrograms (I:C); each dose volume was 50 microliters. Each treatment group contained 6 to 8 mice. Treated mice were sacrificed using CO 2, after the last dose 24 hours, lung cannula. 1 ml of PBS was injected into the lungs for bronchial washing (BAL), and the effluent was recovered. The BAL preparations were then centrifuged to pellet the cells, and the cell-free supernatant was collected and stored at -80 °C for multi-channel cytokine assays. All assessments are based on established guidelines for animal care and use.
酌情使用Luminex多通道分析(Luminex Corp.,Austin,TX)及接合IFN γ、IL-1α、IL-6、CXCL10、JE、KC、MGCSF、MIP1α、RANTES、TNFα、或GMCSF專一單株抗體之珠粒(LINCO Research,St.Charles,MO)測定BAL上澄液中之細胞介素含量。各細胞介素之Luminex分析法係根據廠商指示進行。數據以得自6至8隻小鼠的平均微微克/毫升±平均值之標準誤差(SEM)表示。Use Luminex as appropriate Multichannel analysis (Luminex Corp., Austin, TX) and beads conjugated to IFN γ, IL-1α, IL-6, CXCL10, JE, KC, MGCSF, MIP1α, RANTES, TNFα, or GMCSF specific antibodies (LINCO) Research, St. Charles, MO) determined the interleukin content in BAL. Luminex of each interleukin The analysis method is based on the manufacturer's instructions. Data are expressed as the standard error (SEM) of the average picogram per milliliter ± mean from 6 to 8 mice.
結果指示,50或100微克多I:C之多次投與誘發升高之細胞介素、化學激素及生長因子包括干擾素γ(IFN γ)、介白素-6(IL-6)、組織壞死因子-α(TNFα)、化學激素(CXC主結構)配位體10(CXCL10)、化學激素(CC主結構)配位體2(JE)、化學激素KC(KC)、巨噬細胞炎性蛋白-1α(MIP-1α)、根據活化作用調控、正常地T細胞表現及分泌/CCL5(RANTES)、鼠類顆粒細胞群落刺激因子(mG-CSF)及顆粒細胞-巨噬細胞群落-刺激因子(GM-CSF)等蛋白質含量(表1)。此結果指示,TLR3活化作用可能於細胞介素、化學激素、及生長因子傳介之肺病變例如COPD中扮演重要角色。The results indicate that multiple doses of 50 or 100 micrograms of I:C induce increased levels of interleukins, chemical hormones, and growth factors including interferon gamma (IFN gamma), interleukin-6 (IL-6), tissue. Necrosis factor-α (TNFα), chemical hormone (CXC main structure) ligand 10 (CXCL10), chemical hormone (CC main structure) ligand 2 (JE), chemical hormone KC (KC), macrophage inflammatory Protein-1α (MIP-1α), regulated by activation, normal T cell expression and secretion/CCL5 (RANTES), murine granulosa cell community stimulating factor (mG-CSF), and granulosa cell-macrophage community-stimulator Protein content such as (GM-CSF) (Table 1). This result indicates that TLR3 activation may play an important role in interleukins, chemical hormones, and growth factor-mediated lung lesions such as COPD.
此外,肺組織之Taqman即時PCR分析證明,多次投與引起諸細胞介素基因以及多種TLRs之mRNA及細胞內傳訊分子(表2)之向上調控。彼等數據證明,活體內投與多I:C,一種合成之雙股RNA類似物,引起級聯情況,造成多種促炎性細胞介素、化學激素之分泌及例如TLR2、TLR3、TLR7與TLR9等TLR基因表現之向上調控。In addition, Taqman real-time PCR analysis of lung tissue demonstrated multiple up-regulation of mRNA and intracellular signaling molecules (Table 2) that cause interleukin genes and multiple TLRs. These data demonstrate that in vivo administration of multiple I:C, a synthetic double-stranded RNA analog, causes cascading, resulting in the secretion of various pro-inflammatory interleukins, chemical hormones such as TLR2, TLR3, TLR7 and TLR9 The upregulation of TLR gene expression.
TLR3 活化作用增加肺組織中細胞介素、化學激素、生長因子及鐸基因轉錄量 利用即時PCR(RT-PCR)測定從根據上文實例8所述處理之公或母C57BL/6小鼠之肺中抽取的總RNA之轉錄量。總RNA係使用TrizolT M (Invitrogen Corp.,Carlsbad,CA)自小鼠肺組織試樣抽取,並使用RNEaSy Mini套組(Qiagen Inc.,Valencia,CA)予以單離。然後收集得自進行相同處理的6至8隻小鼠之RNA。 TLR3 activation increases interleukin, chemical hormone, growth factor, and sputum gene transcription in lung tissue. The lungs of male or female C57BL/6 mice treated according to Example 8 above were assayed by real-time PCR (RT-PCR). The amount of total RNA extracted in the transcript. Total RNA was extracted from mouse lung tissue samples using Trizol T M (Invitrogen Corp., Carlsbad, CA) and isolated using the RNEaSy Mini kit (Qiagen Inc., Valencia, CA). RNA from 6 to 8 mice subjected to the same treatment was then collected.
使用OmniscriptT M 套組(Qiagen Inc.,Valencia,CA),根據廠商之操作指南,自各RNA收集物製備cDNAs。如廠商指示,使用TaqManT M Low Density Immune Profiling Array Cards(Applied Biosystems,Foster City,CA)或訂製之Low Density Array(LDA)顯示卡擴增100奈克cDNA。使用Primer ExpressT M 軟體(Applied Biosystems)設計探針與引子組合。然後根據廠商指示,使用ABI PRISMT M 7000HT儀器設備(Applied Biosystems),於384槽版式中進行TaqManT M RT-PCR(Applied Biosystems)。cDNAs were prepared from each RNA collection using an Omniscript T M kit (Qiagen Inc., Valencia, CA) according to the manufacturer's protocol. 100 ng cDNA was amplified using TaqMan T M Low Density Immune Profiling Array Cards (Applied Biosystems, Foster City, CA) or a custom Low Density Array (LDA) display card as indicated by the manufacturer. Probes and primer combinations were designed using Primer Express T M software (Applied Biosystems). TaqMan T M RT-PCR (Applied Biosystems) was then performed in a 384-well format using an ABI PRISM T M 7000HT instrument (Applied Biosystems) according to the manufacturer's instructions.
使用ABI PRISMT M 7000HT儀器設備及相關軟體進行PCR早期指數期之數據收集及轉錄定量。對照18S核糖體RNA之轉錄量,將個別轉錄量常態化。表2中之數據係以相較於使用PBS載劑處理之小鼠,接受多次投與多(I:C)小鼠之mRNA轉錄量平均增加倍數表示。數據代表自6至8隻小鼠收集之RNA。Data collection and transcription quantification of early PCR stages were performed using ABI PRISM T M 7000HT instrumentation and related software. Individual transcripts were normalized against the amount of 18S ribosomal RNA transcription. The data in Table 2 is expressed as the mean increase in mRNA transcription amount of multiple (I:C) mice administered multiple times compared to mice treated with PBS vehicle. Data represent RNA collected from 6 to 8 mice.
數據指出,TLR3活化作用增加鼠類肺組織中細胞介素、化學激素、生長因子及鐸基因轉錄作用(例如TLR3及其他類鐸受體)(表2)。此結果進一步指示,TLR3活化作用及其他類鐸受體(TLRs)之活化作用可能於細胞介素、化學激素、與生長因子傳介之肺病變中扮演重要角色。The data indicate that TLR3 activation increases interleukin, chemical hormone, growth factor, and purine gene transcription in murine lung tissues (eg, TLR3 and other terpenoid receptors) (Table 2). This result further indicates that activation of TLR3 and activation of other terpenoid receptors (TLRs) may play an important role in interleukins, chemical hormones, and lung diseases transmitted by growth factors.
TLR3 活化作用增加肺組織之炎性細胞量 TLR3活化作用增加鼠類肺組織之炎性細胞量(圖13、14、與15)。此結果指示,TLR3活化作用可能於與肺被炎性細胞(圖13)例如嗜中性白血球(圖14)及單核細胞(圖15)(例如單核白血球或淋巴細胞)滲入增加相關之肺病變中扮演重要角色。 Activation of TLR3 increases inflammatory cell mass in lung tissue TLR3 activation increases the amount of inflammatory cells in murine lung tissue (Figures 13, 14, and 15). This result indicates that TLR3 activation may be associated with increased lung infiltration of inflammatory cells (Fig. 13) such as neutrophils (Fig. 13) and monocytes (Fig. 15) (eg, mononuclear leukocytes or lymphocytes). It plays an important role in the lesion.
炎性細胞滲入接受多(I:C)的C57BL/6小鼠肺中之評估係利用血球計數法(圖13)或鑑別染色法(圖14與圖15)進行。小鼠接受如上文實例8所述之多次多(I:C)劑量或單一多(I:C)劑量。單一多(I:C)劑量係經鼻內投與經異佛蘭麻醉之公或母C57BL/6小鼠。所有小鼠介於八至十二週大。單一劑量由50微克或100微克多(I:C)於50微升PBS中組成。BAL回收肺浸潤細胞,接受單一多(I:C)劑量之動物係於多(I:C)投與24小時後進行;接受多劑量之動物則於最後多(I:C)投與24小時後進行。BAL係根據上文實例8所述進行。Evaluation of inflammatory cell infiltration into the lungs of multiple (I:C) C57BL/6 mice was performed using hematocrit (Fig. 13) or differential staining (Fig. 14 and Fig. 15). Mice received multiple multiple (I:C) doses or single multiple (I:C) doses as described in Example 8 above. Single (I:C) doses were administered intranasally to male or female C57BL/6 mice anesthetized with isophora. All mice ranged from eight to twelve weeks old. A single dose consisted of 50 micrograms or more than 100 micrograms (I:C) in 50 microliters of PBS. BAL recovered lung infiltrating cells, and animals receiving a single multi (I:C) dose were administered 24 hours after administration of multiple (I:C); animals receiving multiple doses were administered at the last (I:C) for 24 hours. Afterwards. The BAL was carried out as described in Example 8 above.
使經處理之小鼠肺進行BAL後回收之細胞沉澱物懸浮於含0.1% BSA之200微升杜貝可氏(Dulbecco’s)磷酸鹽緩衝鹽液(DPBS)。然後取50微升該懸浮細胞,將其添加至50微升吐克氏血液(Turk’s Blood)稀釋流體(Red Bird Service,Osgood,IN)中,充分混合,利用血球計計算總細胞數(圖13)。然後取100微升懸浮液(含有少於1 x 105 個細胞/微升)裝填於CytospinT M 載片裝配上,於400 rpm離心4分鐘。自CytospinT M 裝配移出載片,令其乾燥至少一小時。接著將載片浸於Wright-Giemsa中染色90秒,於ddH2 O中脫色5分鐘。令載片乾燥隔夜。使用100倍物鏡,將油鏡浸於油中,進行載片之鑑別計數,計算嗜中性白血球(圖14)與單核細胞(圖15)總數。然後將收集自各處理組別之6至8隻小鼠之肺浸潤細胞平均值與SEM數據製圖(圖13、14、與15)。The cell pellet recovered after BAL treatment of the treated mouse lungs was suspended in 200 microliters of Dulbecco's phosphate buffered saline (DPBS) containing 0.1% BSA. Then, 50 μl of the suspension cells were taken, added to 50 μl of Turk's Blood dilution fluid (Red Bird Service, Osgood, IN), thoroughly mixed, and the total number of cells was counted using a hemocytometer (Fig. 13 ). 100 microliters of the suspension was then taken (containing less than 1 x 10 5 cells / [mu] l) was loaded on the carrier sheet assembly Cytospin T M, centrifuged at 400 rpm for 4 minutes. The slides were removed from the Cytospin T M assembly and allowed to dry for at least one hour. The slides were then immersed in Wright-Giemsa for 90 seconds and destained in ddH 2 O for 5 minutes. Let the slides dry overnight. Using a 100x objective, the oil mirror was immersed in oil, and the identification of the slides was counted to calculate the total number of neutrophils (Fig. 14) and monocytes (Fig. 15). The mean values of lung infiltrating cells collected from 6 to 8 mice from each treatment group were then plotted against SEM data (Figures 13, 14, and 15).
TLR3 基因剔除動物免除肺組織中由多 (I:C) 誘發的炎性細胞量之增加 利用血球計數法及鑑別染色法鑑定嗜中性白血球與單核細胞,以評估炎性細胞之滲入C57BL/6或TLR3基因剔除小鼠或接受單一或多次多(I:C)投與小鼠之肺中。小鼠如實例8所述接受多次多(I:C)劑量或如實例10所述接受單一多(I:C)劑量。BAL回收肺浸潤細胞,接受單一多(I:C)劑量之動物係於多(I:C)投與24小時後進行;接受多劑量之動物則於最後多(I:C)投與24小時後進行。BAL係根據上文實例8所述進行。如實例10所述,利用血球計數法或鑑別染色法評估炎性細胞之滲入野生型C57BL/6或TLR3基因剔除小鼠之肺中。數據以相較於單獨接受PBS動物之平均肺浸潤細胞計數,多(I:C)處理動物平均肺浸潤細胞計數之增加倍數表示。數據代表得自6隻小鼠之值。 TLR3 knockout animals exempted lung tissue from increased (I:C) -induced increase in inflammatory cells. Hematocrit and differential staining were used to identify neutrophils and monocytes to assess the infiltration of inflammatory cells into C57BL/ 6 or TLR3 knockout mice or one or more multiple (I:C) administered to the lungs of mice. Mice received multiple multiple (I:C) doses as described in Example 8 or received a single multiple (I:C) dose as described in Example 10. BAL recovered lung infiltrating cells, and animals receiving a single multi (I:C) dose were administered 24 hours after administration of multiple (I:C); animals receiving multiple doses were administered at the last (I:C) for 24 hours. Afterwards. The BAL was carried out as described in Example 8 above. Infiltration of inflammatory cells into the lungs of wild-type C57BL/6 or TLR3 knockout mice was assessed by hemocytometry or differential staining as described in Example 10. Data are expressed as a fold increase in the mean lung infiltrating cell count of multiple (I:C) treated animals compared to the mean lung infiltrating cell count of PBS animals alone. Data represent values from 6 mice.
表3所示結果指出,相較於野生型小鼠,TLR3基因剔除小鼠免除肺組織中由多(I:C)誘發的炎性細胞量之增加;多(I:C)投與之影響主要係由於TLR3活化作用。再者,結果指示,TLR3活化作用可能於與肺被炎性細胞例如嗜中性白血球及單核細胞(例如單核白血球或淋巴細胞)滲入增加相關之肺病變中扮演重要角色。The results shown in Table 3 indicate that TLR3 knockout mice exempt the increase in the amount of inflammatory cells induced by multiple (I:C) in lung tissue compared to wild-type mice; the effect of multiple (I:C) administration Mainly due to TLR3 activation. Furthermore, the results indicate that TLR3 activation may play an important role in lung lesions associated with increased lung infiltration by inflammatory cells such as neutrophils and monocytes such as mononuclear leukocytes or lymphocytes.
表 3:
相較於野生型小鼠,TLR3基因剔除(KO)小鼠免除肺組織中由多(I:C)誘發的炎性細胞量之增加。數據以相較於單獨接受PBS動物之平均肺浸潤細胞計數,多(I:C)處理動物平均肺浸潤細胞計數之增加倍數表示。數據代表得自6隻小鼠之值。
具有多 (I:C) 之 TLR3 活化作用於乙醯甲基膽鹼攻毒動物中進一步損害肺功能 每24小時,使野生型公或母C57BL/6小鼠接受單一多(I:C)劑量於PBS中或單獨PBS(圖16)或多(I:C)於PBS中之三種劑量或單獨PBS之鼻內投與三天(圖17)。多(I:C)活化TLR3。所有小鼠均為十二週大。各多(I:C)劑量含有50微克或100微克多(I:C),容積為50微升。各處理組別含6至8隻小鼠。 Having a plurality of (I: C) of acetyl TLR3 activation on methacholine challenge is further lung damage in animal every 24 hours, wild-type male or female C57BL / 6 mice received a single multiple (I: C) Doses were administered intranasally in PBS or three doses of PBS alone (Figure 16) or multiple (I:C) in PBS or PBS alone for three days (Figure 17). Multiple (I:C) activates TLR3. All mice were twelve weeks old. Each multi (I:C) dose contained 50 micrograms or more than 100 micrograms (I:C) with a volume of 50 microliters. Each treatment group contained 6 to 8 mice.
使用PenH值作為呼吸道阻塞標記及最後一次多(I:C)劑量24小時後之喘息效應(breathing effect)評估肺功能。如圖16或圖17所示,利用全身體積變化描記器(WBP),自增加暴露於乙醯甲基膽鹼攻毒之小鼠收集PenH值。乙醯甲基膽鹼增加喘息效應且損害肺功能。使乙醯甲基膽鹼溶於PBS中,呈噴霧氣溶膠投與。所有評估均根據已建立之動物照料及使用指導方針進行。圖16與17中之數據代表得自各處理組別6至8隻小鼠之平均值與SEM。Pulmonary function was assessed using the PenH value as the airway obstruction marker and the breathing effect after 24 hours of the last multiple (I:C) dose. As shown in Figure 16 or Figure 17, PenH values were collected from mice challenged with exposure to ethylene choline challenge using a whole body volume change spirograph (WBP). Ethyl choline increases the wheezing effect and impairs lung function. Ethyl choline was dissolved in PBS and administered as a spray aerosol. All assessments are based on established guidelines for animal care and use. The data in Figures 16 and 17 represent the mean and SEM of 6 to 8 mice from each treatment group.
結果指示,TLR3之活化進一步於乙醯甲基膽鹼攻毒野生型小鼠中損害肺功能(圖16與圖17)。此結果暗示,TLR3活化作用可能進一步損害由於感染、慢性阻塞性肺疾(COPD)、或其他疾病已受肺傷害之苦的個體之肺功能。因此,拮抗TLR3活性之治療處置可防止已受損害肺功能之苦的個體附加之肺功能傷害。The results indicated that activation of TLR3 further impaired lung function in challenged wild-type mice with acetaminophen (Figure 16 and Figure 17). This result suggests that TLR3 activation may further impair lung function in individuals who have suffered from lung injury due to infection, chronic obstructive pulmonary disease (COPD), or other diseases. Thus, therapeutic treatments that antagonize TLR3 activity prevent additional lung function damage in individuals who have suffered from impaired lung function.
TLR3 基因剔除動物於乙醯甲基膽鹼攻毒期間免受由多 (I:C) 誘發之肺功能傷害 使如實例12所述之公或母野生型C57BL/6小鼠或TLR3基因剔除小鼠進行單一(圖18)及多劑量(圖19)多(I:C)投與。使用利用如實例12所述WBP收集之PenH值評估肺功能。乙醯甲基膽鹼投與亦如實例12所述。所有評估均根據已建立之動物照料及使用指導方針進行。圖18與19中之數據代表得自各處理組別6至8隻小鼠之平均值與SEM。 TLR3 knockout animals were protected from multiple (I:C) -induced lung function damage during challenge with methotrexate methylcholine . The male or female wild-type C57BL/6 mice or TLR3 gene knockouts as described in Example 12 were small. Rats were administered a single (Figure 18) and multiple dose (Figure 19) multiple (I:C) administration. Lung function was assessed using PenH values collected using WBP as described in Example 12. Ethyl choline administration was also as described in Example 12. All assessments are based on established guidelines for animal care and use. The data in Figures 18 and 19 represent the mean and SEM of 6 to 8 mice from each treatment group.
TLR3基因剔除動物於乙醯甲基膽鹼攻毒期間免受由多(I:C)誘發之肺功能傷害(圖18與圖19)。此結果指示,括抗TLR3活性之治療處置可防止由於感染、慢性阻塞性肺疾(COPD)、或其他疾病已受肺功能傷害之苦的個體附加之肺功能傷害。此外,此結果進一步指示,多(I:C)投與之影響主要由於TLR3活化作用。TLR3 knockout animals were protected from multiple (I:C)-induced lung function damage during challenge with methotrexate methylcholine (Figure 18 and Figure 19). This result indicates that treatment treatment including anti-TLR3 activity prevents additional lung function damage in individuals suffering from lung function damage due to infection, chronic obstructive pulmonary disease (COPD), or other diseases. Furthermore, this result further indicates that the effect of multiple (I:C) administration is mainly due to TLR3 activation.
hTLR3 拮抗劑對人類肺支氣管上皮細胞中細胞介素與化學激素產生之影響 人類肺支氣管上皮細胞株BEAS-2B係得自美國菌種保存中心(CRL-9609)。使BEAS-2B生長於被覆膠原蛋白I燒瓶(BD Biosciences)之LHC-9不含血清培養基中,於0.25%胰蛋白酶/EDTA中簡略洗滌後,予以收取。然後於LHC-9不含血清培養基(Biosource)中洗滌細胞,以1x106 /毫升之濃度使其再懸浮於LHC-9培養基中。將細胞塗佈於被覆膠原蛋白I之96槽平底盤上,每槽200微升;每一條件進行三重複培養槽。 Effect of hTLR3 antagonist on interleukin and chemical hormone production in human lung bronchial epithelial cells The human lung bronchial epithelial cell line BEAS-2B was obtained from the American Type Culture Collection (CRL-9609). BEAS-2B was grown in LHC-9 serum-free medium in a coated collagen I flask (BD Biosciences), and washed briefly after washing in 0.25% trypsin/EDTA. The cells were then washed in LHC-9 serum-free medium (Biosource) and resuspended in LHC-9 medium at a concentration of 1 x 10 6 /ml. The cells were plated onto a 96-well flat-bottomed plate coated with collagen I at 200 microliters per well; three replicate culture tanks were performed for each condition.
經6小時培育令細胞附著後,取出培養基,以200微升新鮮培養基替換。每槽添加125奈克TLR3促效劑多(I:C)之前,於37℃培育mAb 1068之十倍系列稀釋液40分鐘。經多(I:C)刺激24小時後,收集培養上澄液,進行試樣之Luminex多通道分析(Luminex Corp.,Austin,TX)以檢測IL-6、IL-8、RANTES、MCP-1、IP-10、IFN-α、IFN-γ、IL-1β、IL-12、TNF-α、MCP-1、與IL-10表現量。After 6 hours of incubation to allow cells to attach, the medium was removed and replaced with 200 microliters of fresh medium. A ten-fold serial dilution of mAb 1068 was incubated at 37 °C for 40 minutes before adding 125 ng of TLR3 agonist (I:C) per well. After 24 hours of stimulation with multiple (I:C), the culture supernatant was collected and the sample was subjected to Luminex. Multichannel analysis (Luminex Corp., Austin, TX) to detect IL-6, IL-8, RANTES, MCP-1, IP-10, IFN-α, IFN-γ, IL-1β, IL-12, TNF- The amount of α, MCP-1, and IL-10 expression.
結果指示,抗-TLR3拮抗劑mAb 1068(與圖20之mAb CNTO260相同)於多(I:C)刺激之BEAS-2B細胞中降低IL-6、IL-8、RANTES、MCP-1與IP-10產生。如圖20所示,IL-6、IL-8、RANTES、MCP-1與IP-10之表現以mAb 1068劑量依存方式被降低。試樣中未檢測到IFN-α、IFN-γ、IL-1β、IL-12、TNF-α、MCP-1、與IL-b表現。The results indicate that the anti-TLR3 antagonist mAb 1068 (identical to the mAb CNTO260 of Figure 20) reduces IL-6, IL-8, RANTES, MCP-1 and IP- in multiple (I:C) stimulated BEAS-2B cells. 10 produced. As shown in Figure 20, the performance of IL-6, IL-8, RANTES, MCP-1 and IP-10 was reduced in a dose-dependent manner of mAb 1068. No expression of IFN-α, IFN-γ, IL-1β, IL-12, TNF-α, MCP-1, and IL-b was detected in the sample.
hTLR3 拮抗劑處理增加致命性肺炎之存活 於彼等實驗中,8至10週大之野生型C57BL/6母小鼠,以於50微升PBS中之5個斑點形成單位(PFU)流行性感冒病毒A/PR/8經鼻內予以感染,接著於8天後,以於50微升PBS中之50個群落形成單位(CFU)肺炎鏈球菌經鼻內予以感染。 hTLR3 antagonist treatment increased the survival of fatal pneumonia in their experiments, 8 to 10 weeks old wild-type C57BL/6 female mice, 5 spots forming units (PFU) influenza in 50 microliters of PBS Virus A/PR/8 was infected intranasally, followed by intranasal infection with 50 colony forming units (CFU) of S. pneumoniae in 50 microliters of PBS after 8 days.
該病毒與細菌劑量單獨投與時未達致死量,惟彼等劑量一起,則對大多數小鼠具致命性(圖22)。模擬感染之對照組小鼠接受PBS而非流行性感冒病毒A/PR/8或肺炎鏈球菌。hTLR3拮抗劑處理小鼠於第8天接種肺炎鏈球菌之前2小時,經由腹膜內注射接受0.6毫克或0.06毫克(於0.2毫升PBS中)之投與(預防性投與),於第9天再同法進行投與(治療性投與)。對照組模擬處理小鼠經由腹膜內投與,接受0.6毫克或0.06毫克於PBS中之非專一性IgG。各處理或對照組含7隻小鼠。此處所述之所有評估均根據已建立之動物照料及使用指導方針進行。The virus did not reach lethal dose when administered alone with the bacterial dose, but together with the doses, it was lethal to most mice (Figure 22). Control infected mice received PBS instead of influenza virus A/PR/8 or S. pneumoniae. hTLR3 antagonist-treated mice received 2 mg of S. pneumoniae on day 8 and received 0.6 mg or 0.06 mg (in 0.2 ml of PBS) via intraperitoneal injection (prophylactic administration), and on day 9 again The same method of administration (therapeutic investment). The control mock-treated mice were administered intraperitoneally and received 0.6 mg or 0.06 mg of non-specific IgG in PBS. Each treatment or control group contained 7 mice. All assessments described herein are based on established animal care and use guidelines.
於雞蛋中培養流行性感冒A/PR/8病毒,使用具有MDCK細胞之標準分析法測定PFU力價,並呈供接種用之凍結病毒貯存株保存。於含5%綿羊血之胰化酪蛋白大豆洋菜培養皿(TSA/血液)上,使肺炎鏈球菌(ATCCNumber:6301T M )種菌生長隔夜,然後從培養皿中移出細菌,使其懸浮於磷酸鹽緩衝鹽液(PBS)中。使用600奈米處之光密度及標準方法計算PBS懸浮液中之細菌CFU力價。接著於PBS中製備細菌種菌。利用標準群落形成試驗確定細菌種菌中之CFU,以測定投與小鼠之種菌中實際存在之細菌數。The influenza A/PR/8 virus was cultured in eggs, and the PFU price was measured using a standard assay method for MDCK cells, and stored as a frozen virus stock for inoculation. Streptococcus pneumoniae (ATCC) on a yeast casein soy dish (TSA/blood) containing 5% sheep blood Number: 6301 T M ) The inoculum grows overnight, then the bacteria are removed from the culture dish and suspended in phosphate buffered saline (PBS). The bacterial CFU price in the PBS suspension was calculated using the optical density at 600 nm and standard methods. Bacterial inoculum was then prepared in PBS. The CFU in the bacterial inoculum was determined using a standard colony formation assay to determine the number of bacteria actually present in the inoculum administered to the mice.
製備種菌後,如上文所述使用流行性感冒A/PR/8病毒或肺炎鏈球菌經鼻內感染小鼠。模擬感染之對照組小鼠如上文所述接受經鼻內投與之PBS。hTLR3拮抗劑處理小鼠如上文所述接受經腹膜內預防性及治療性投與之mAb 1068。模擬處理之對照組小鼠如上文所述接受經腹膜內投與之於PBS中之非專一性IgG。單獨流行性感冒A/PR/8病毒與肺炎鏈球菌劑量未達致死量,因為被單獨病毒或細菌感染之小鼠100%存活(圖22)。然而,彼等用它法未達致死劑量之病毒或細菌加在一起感染,則使大多數小鼠產生致命性肺炎(圖22)。After the inoculum was prepared, the mice were intranasally infected with influenza A/PR/8 virus or S. pneumoniae as described above. Control infected mice were treated with intranasal administration of PBS as described above. hTLR3 antagonist treated mice received intraperitoneal prophylactically and therapeutically administered mAb 1068 as described above. The mock-treated control mice received non-specific IgG administered intraperitoneally in PBS as described above. Influenza A/PR/8 virus alone and S. pneumoniae doses did not reach lethal doses because mice infected with virus alone or bacteria were 100% viable (Figure 22). However, they were infected with viruses or bacteria that did not reach a lethal dose, resulting in fatal pneumonia in most mice (Figure 22).
細菌感染48小時後,使小鼠安樂死,無菌操作下獲取肺臟,於無菌PBS中均質化,於PBS中製備均漿稀釋液,將稀釋液放置於TSA/血液培養皿上,以測定肺臟中之細菌負擔。然後培育該等培養皿至看見菌落並計算CFUs。如圖23所示,先前以未達致死劑量之流行性感冒病毒感染,經肺炎鏈球菌感染2天後,增加肺中之細菌負擔。After 48 hours of bacterial infection, the mice were euthanized, the lungs were aseptically obtained, homogenized in sterile PBS, and the homogenate dilution was prepared in PBS, and the dilution was placed on a TSA/blood culture dish to determine the lungs. Bacterial burden. The dishes were then incubated until colonies were seen and CFUs were calculated. As shown in Figure 23, the infection with an influenza virus that did not reach a lethal dose was previously increased by 2 days after infection with S. pneumoniae, increasing the burden of bacteria in the lungs.
相較於接受0.6毫克或0.06毫克非專一IgG對照組單株抗體之對照組小鼠,於第8及第9天每隻小鼠投與0.6毫克或0.06毫克抗-TLR3 mAb 1068增加被流行性感冒病毒A/PR/8與肺炎鏈球菌感染的小鼠之存活率(圖22)。Compared with control mice receiving 0.6 mg or 0.06 mg of non-specific IgG control monoclonal antibody, each mouse was given 0.6 mg or 0.06 mg anti-TLR3 mAb 1068 on Days 8 and 9 to increase the prevalence. Survival rates of cold virus A/PR/8 and mice infected with S. pneumoniae (Figure 22).
重要的是,C57BL/6母小鼠平均體重介於18克與20克之間;因此對接受0.6毫克mAb 1068之小鼠而言,所投與TLR3拮抗劑之劑量範圍為大約3.0毫克/公斤與3.3毫克/公斤體重之間,接受0.06毫克mAb 1068之小鼠則為大約30毫克/公斤與33毫克/公斤體重之間。圖21係標示此範圍之較低端。Importantly, the average body weight of C57BL/6 female mice ranged from 18 grams to 20 grams; therefore, for mice receiving 0.6 mg mAb 1068, the dose administered to the TLR3 antagonist ranged approximately 3.0 mg/kg. Between 3.3 mg/kg body weight, mice receiving 0.06 mg mAb 1068 were between approximately 30 mg/kg and 33 mg/kg body weight. Figure 21 shows the lower end of this range.
TLR3 活性對結腸上皮細胞增殖率之影響 於鼠類模式中,結腸上皮細胞之增殖率由於剔除TLR3受體基因活性而增加(數據示於表4)。於彼等實驗中,如上文所述之野生型C57BL/6公小鼠或TLR3基因剔除小鼠各自經腹膜內給與於1毫升PBS中之1毫克溴脫氧尿苷(BrdU),2小時後,犧牲小鼠。所有小鼠為6至8週大,各處理組別具有至少3隻小鼠。 Effect of TLR3 activity on colonic epithelial cell proliferation rate In the murine model, the proliferation rate of colonic epithelial cells increased due to knockout of TLR3 receptor gene activity (data shown in Table 4). In these experiments, wild-type C57BL/6 male mice or TLR3 knockout mice as described above were intraperitoneally administered with 1 mg of bromodeoxyuridine (BrdU) in 1 ml of PBS, 2 hours later. , sacrificing mice. All mice were 6 to 8 weeks old and each treatment group had at least 3 mice.
然後獲取供組織病理學分析用之結腸。將結腸組織固定,切段,包埋於石蠟中,製備5微米之切片。根據廠商之操作指南,使切片相繼與小鼠抗-BrdU IgG單株抗體(Becton-Dickinson Biosciences,Inc.,San Jose,CA)、山羊抗-小鼠IgG單株抗體(Becton-Dickinson Biosciences,Inc.,San Jose,CA)、山葵過氧化酶(HRP)接合物(Becton-Dickinson Biosciences,Inc.,San Jose,CA)、及二胺基聯苯胺(DAB)基質(Becton-Dickinson Biosciences,Inc.,San Jose,CA)培育。培育後之切片使用標準方法,以蘇木素進行對比染色。The colon for histopathological analysis is then obtained. Colon tissue was fixed, sectioned, embedded in paraffin, and 5 micron sections were prepared. Sections were sequentially subjected to mouse anti-BrdU IgG monoclonal antibody (Becton-Dickinson Biosciences, Inc., San Jose, CA), goat anti-mouse IgG monoclonal antibody (Becton-Dickinson Biosciences, Inc.) according to the manufacturer's instructions. ., San Jose, CA), Wasabi Peroxidase (HRP) conjugate (Becton-Dickinson Biosciences, Inc., San Jose, CA), and diaminobenzidine (DAB) matrix (Becton-Dickinson Biosciences, Inc. , San Jose, CA). The sections after incubation were subjected to comparative staining with hematoxylin using standard methods.
然後直觀審查經培育之切片,計算結腸腺窩中因BrdU併入DNA中而呈陽性染色之細胞數。於得自相同片段的切片之24個妥為排列之延續腺窩中計算細胞。以BrdU併入作為鑑定經由細胞週期進展的細胞(亦即,增殖細胞)之代用標記。表4中,增殖率數據以每2小時每隻動物每一結腸腺窩BrdU染色細胞之平均數表示。彼等數據以平均增殖率±標準偏差(P<0.0001,T-試驗)呈現。The cultured sections were then visually reviewed and the number of cells stained positive for BrdU in the colon gland was calculated. Cells were counted in 24 appropriately arranged colons from the same fragment. BrdU is incorporated as a surrogate marker for identifying cells that progress through the cell cycle (i.e., proliferating cells). In Table 4, the proliferation rate data is expressed as the average number of BrdU-stained cells per colon of each animal per 2 hours. Their data were presented as mean proliferation rate ± standard deviation (P < 0.0001, T-test).
數據指示,TLR3之不活化作用增加結腸上皮細胞之增殖作用。The data indicates that the inactivation of TLR3 increases the proliferation of colonic epithelial cells.
TLR3 活性於炎性腸道疾病復原期間對結腸上皮細胞增殖率之影響 於鼠類模式炎性腸道疾病(IBD)復原期間,結腸上皮細胞之增殖率由於剔除TLR3受體基因活性而增加(表5)。 Effect of TLR3 activity on the proliferation rate of colonic epithelial cells during the recovery of inflammatory bowel disease During the recovery of murine model inflammatory bowel disease (IBD), the proliferation rate of colonic epithelial cells increases due to knockout of TLR3 receptor gene activity (Table) 5).
於彼等實驗中,野生型C57BL/6公小鼠或上述TLR3 KO小鼠各給與含5%(w/v)硫酸鈉葡聚醣(DSS)之飲用水3天,以誘發急性潰瘍性結腸炎。然後供給白水直至30小時後實驗終了。於開始接受白水6小時後,如上文所述,對小鼠注射BrdU。然後令小鼠自DSS誘發的潰瘍性結腸炎復原24小時後,犧牲小鼠。所有小鼠為6至8週大,各處理組別具有至少3隻小鼠。In these experiments, wild type C57BL/6 male mice or the above TLR3 KO mice were given drinking water containing 5% (w/v) sodium sulfate dextran (DSS) for 3 days to induce acute ulcerativeness. colitis. White water was then supplied until the end of the experiment after 30 hours. After 6 hours of receiving white water, the mice were injected with BrdU as described above. The mice were then sacrificed 24 hours after DSS-induced ulcerative colitis, and the mice were sacrificed. All mice were 6 to 8 weeks old and each treatment group had at least 3 mice.
如上文實例15所述製備及分析供結腸腺窩細胞增殖之組織病理學分析用之結腸試樣。增殖率數據以每24小時每隻動物每一結腸腺窩BrdU染色細胞之平均數表示。彼等數據以平均增殖率±標準偏差(P<0.004,T-試驗)呈現。表5之數據指示,於炎性腸道疾病復原期間,TLR3之不活化作用增加結腸上皮細胞之增殖率。Colon samples for histopathological analysis of colonic cell proliferation were prepared and analyzed as described in Example 15 above. Proliferation data are expressed as the average number of BrdU-stained cells per colon of each animal per 24 hours. Their data are presented as mean proliferation rate ± standard deviation (P < 0.004, T-test). The data in Table 5 indicates that the inactivation of TLR3 increases the proliferation rate of colonic epithelial cells during the recovery of inflammatory bowel disease.
TLR3 基因剔除小鼠之胰島素敏感性 TLR3剔除(KO)(C57BL/6背景)及野生型(WT)對照組小鼠(C57 Bl/6)以由60.9%千卡脂肪與20.8%千卡碳水化合物組成之高脂飼料(Purina TestDiet #58126)餵飼。對照組TLR3 KO及WT小鼠以正常飼料餵飼。使動物禁食隔夜,經由腹膜內注射1.0毫克/克葡萄糖以進行葡萄糖耐受性試驗(GTT),於0、15、30、60、90、及120分鐘取得血糖讀數。 TLR3 knockout mice with insulin-sensitive TLR3 knockout (KO) (C57BL/6 background) and wild-type (WT) control mice (C57 Bl/6) with 60.9% kcal fat and 20.8% kcal carbohydrate The high fat feed (Purina TestDiet #58126) was fed. The control group TLR3 KO and WT mice were fed with normal diet. Animals were fasted overnight, and a glucose tolerance test (GTT) was performed by intraperitoneal injection of 1.0 mg/g glucose for glucose readings at 0, 15, 30, 60, 90, and 120 minutes.
圖31顯示,相較於高脂飼料餵飼之野生型小鼠,高脂飼料餵飼14及26週之TLR3 KO小鼠顯示於葡萄糖耐受性試驗中有改善。餵飼正常飼料之小鼠如預期未展示任何變化。彼等結果顯示,TLR3傳訊作用可能影響胰島素敏感性而提供使用TLR3拮抗劑治療2型糖尿病之基礎。Figure 31 shows that TLR3 KO mice fed a 14- and 26-week high-fat diet showed improvement in the glucose tolerance test compared to wild-type mice fed a high-fat diet. Mice fed normal diet did not show any changes as expected. These results suggest that TLR3 signaling may influence insulin sensitivity and provide a basis for the treatment of type 2 diabetes with TLR3 antagonists.
圖32顯示高脂餵飼及正常飼料餵飼的小鼠之空腹血糖量。相較於高脂飼料餵飼之野生型小鼠,TLR3 KO動物之空腹血糖量常態化。彼等數據暗示,TLR3傳訊作用可能干擾肝臟葡萄糖代謝,促成葡萄糖耐受性不良及胰島素抗阻之進展。Figure 32 shows the fasting blood glucose levels of mice fed with high fat and normal feed. The fasting blood glucose levels of TLR3 KO animals were normalized compared to wild-type mice fed with high fat diet. These data suggest that TLR3 signaling may interfere with hepatic glucose metabolism, contributing to glucose tolerance and progression of insulin resistance.
接著,評估以正常飼料或高脂飼料餵飼的TLR3 KO及野生型小鼠之胰島素量。測量禁食隔夜小鼠於葡萄糖攻毒前後血中胰島素含量。使用Crystal Chem(Downers Grove,IL)Ultra-Sensitive ELISA分析試驗套組(cat # 90060)定量胰島素。以高脂飼料餵飼之TLR3 KO小鼠於基線(未進行葡萄糖攻毒)及葡萄糖攻毒20與60分鐘後,顯示胰島素含量增加(圖33)。整體而言,加上葡萄糖耐受性試驗所得結果,由數據顯示,缺乏TLR3傳訊作用影響胰島素含量及敏感性。Next, the amount of insulin in TLR3 KO and wild type mice fed with normal or high fat diets was evaluated. The insulin content in the blood of fasting mice before and after glucose challenge was measured. The test kit (cat #90060) was used to quantify insulin using a Crystal Chem (Downers Grove, IL) Ultra-Sensitive ELISA assay kit. TLR3 KO mice fed a high-fat diet showed an increase in insulin content at baseline (no glucose challenge) and 20 and 60 minutes after glucose challenge (Figure 33). Overall, coupled with the results of the glucose tolerance test, data showed that the lack of TLR3 signaling affected insulin content and sensitivity.
犧牲以高脂飼料餵飼30週之TLR3 KO小鼠,測定其血清試樣中之脂質概況:測定總膽固醇、HDl、LDL、三酸甘油酯與FFA。簡言之,所有脂質試驗均使用GEMCAL Reference Serum(Alfa Wassermann Diagnostic Technologies,LCC,West Caldwell,NJ),將未知試樣之吸收變化參照標準吸收變化予以校正。每天於記述結果之前,先進行兩個標準對照組。裝填試樣,獲得脂質數據,以習知單位毫克/分升表示。FFA含量係使用NEFA套組(Wako)予以測定。相較於同法餵飼之野生型小鼠,TLR3 KO動物顯示較低之膽固醇、LDL與HDL以及FFA流通量。彼等結果顯示,缺乏TLR3傳訊作用在降低膽固醇量上扮演有利角色,證明TLR3拮抗劑單株抗體於治療心血管疾病及預防與2型糖尿病相關的心血管併發症上之效用。TLR3 KO mice fed a high-fat diet for 30 weeks were sacrificed and lipid profiles in serum samples were determined: total cholesterol, HD1, LDL, triglycerides and FFA were determined. Briefly, all lipid assays were corrected using GEMCAL Reference Serum (Alfa Wassermann Diagnostic Technologies, LCC, West Caldwell, NJ) with reference to changes in absorption of unknown samples. Two standard controls were performed daily before the results were recorded. The sample is loaded and lipid data is obtained, expressed in conventional units of milligrams per deciliter. The FFA content was determined using a NEFA kit (Wako). TLR3 KO animals showed lower cholesterol, LDL and HDL, and FFA flux compared to wild-type mice fed the same method. These results show that the lack of TLR3 signaling plays a beneficial role in lowering cholesterol levels, demonstrating the utility of TLR3 antagonist monoclonal antibodies in the treatment of cardiovascular disease and prevention of cardiovascular complications associated with type 2 diabetes.
總之,所呈現結果顯示,相較於野生型小鼠,以高脂飼料餵飼之TLR3 KO小鼠防禦葡萄糖耐受性不良(為胰島素抗阻之特徵)之進展,證明缺乏TLR3傳訊作用可防護小鼠免受II型糖尿病傷害。再者,數據顯示,相較於以高脂飼料餵飼之野生型小鼠,以高脂飼料餵飼之TLR3 KO小鼠具有較低量之總膽固醇、LDH與HDL膽固醇以及HDLc/LDLc比率,因此表明TLR3拮抗劑於向下調節與心血管疾病相關的危險因子上之有利角色。彼等發現暗示TLR3抑制劑於2型糖尿病、血脂失調及代謝症候群治療方法上之用途。In conclusion, the results showed that TLR3 KO mice fed with high-fat diets were resistant to poor glucose tolerance (characterized by insulin resistance) compared to wild-type mice, demonstrating the lack of TLR3 signaling to protect against Mice are protected from type 2 diabetes. Furthermore, the data showed that TLR3 KO mice fed a high-fat diet had lower amounts of total cholesterol, LDH and HDL cholesterol, and HDLc/LDLc than wild-type mice fed a high-fat diet. This demonstrates the beneficial role of TLR3 antagonists in down-regulating risk factors associated with cardiovascular disease. They found the use of TLR3 inhibitors in the treatment of type 2 diabetes, dyslipidemia and metabolic syndrome.
適應人類之抗 -TLR3 單株抗體之產生及鑑定 使用鼠類抗-TLR3 mAb C1068之胺基酸序列探詢自公共抗體序列資料庫匯編之人類抗體資料庫。C1068(SEQ ID NO:6)之重鏈可變區顯示與四個重鏈胚細胞序列(亦即人類VH1重鏈族之VB_1-03/JH1 72、VB_1-02/JH1 71、VB_1-08/JH1 71與VB_1-69/JH2 70)之最高同質性。於是乃合成四個核酸構築體,其中係將C1068重鏈之諸CDR區域移入所選定人類胚細胞重鏈序列之內,以產生具有分別如SEQ ID NOs:25、27、29及31所示可變區胺基酸序列之四個適應人類之抗-TLR3單株抗體重鏈,命名HV1、HV4、HV5與HV7。 Generation and identification of anti- TLR3 monoclonal antibodies adapted to humans The human antibody library compiled from the public antibody sequence library was probed using the amino acid sequence of murine anti-TLR3 mAb C1068. The heavy chain variable region of C1068 (SEQ ID NO: 6) is shown with four heavy chain blast sequences (ie, human VH1 heavy chain family VB_1-03/JH1 72, VB_1-02/JH1 71, VB_1-08/ The highest homogeneity of JH1 71 and VB_1-69/JH2 70). Thus, four nucleic acid constructs are synthesized in which the CDR regions of the C1068 heavy chain are transferred into the selected human blast cell heavy chain sequence to produce the sequences shown in SEQ ID NOs: 25, 27, 29 and 31, respectively. The four amino acid sequences of the variable region amino acid sequence are adapted to human anti-TLR3 monoclonal antibody heavy chains, and are named HV1, HV4, HV5 and HV7.
C1068(SEQ ID NO:16)之輕鏈可變區顯示與四個輕鏈胚細胞序列(亦即人類VK I族之VB_O12/JK2 78、VB_A30/JK2 77、VB_A20/JK4 76與VB_L1/JK2 76)之最高同質性。於是乃合成四個核酸構築體,其中係將C1068輕鏈之諸CDR區域移入所選定人類胚細胞輕鏈序列之內,以產生具有分別如SEQ ID NOs:33、35、37及39所示可變區胺基酸序列之四個適應人類之抗-TLR3單株抗體輕鏈,命名LV1、LV3、LV5與LV7。The light chain variable region of C1068 (SEQ ID NO: 16) is shown with four light chain germ cell sequences (ie, human VK I family of VB_O12/JK2 78, VB_A30/JK2 77, VB_A20/JK4 76 and VB_L1/JK2 76). The highest homogeneity. Thus, four nucleic acid constructs are synthesized in which the CDR regions of the C1068 light chain are transferred into the selected human embryonic cell light chain sequence to produce as shown in SEQ ID NOs: 33, 35, 37 and 39, respectively. The four amino acid sequences of the variable region amino acid sequence are adapted to human anti-TLR3 monoclonal antibody light chain, and LV1, LV3, LV5 and LV7 are named.
表現代表四個重鏈與四個輕鏈可變區構築體之所有可能組合之十六個單株抗體。所有重鏈可變區架構以全長重鏈中殘基108之Ser替換為Pro,Phe114與Leu115替換為Ala(SEQ ID NO:41);S228P、F234A及L235A之人類IgG4重鏈恒定區表現。所有輕鏈可變區架構以人類K恒定區(SEQ ID NO:4)表現。Sixteen individual antibodies representing all possible combinations of four heavy chain and four light chain variable region constructs. All heavy chain variable region architectures were replaced with Ser of residue 108 in the full length heavy chain by Pro, Phe114 and Leu115 replaced by Ala (SEQ ID NO: 41); human IgG4 heavy chain constant regions of S228P, F234A and L235A. All light chain variable region architectures are represented by the human K constant region (SEQ ID NO: 4).
利用含有質體的適當重鏈與輕鏈之共轉染,於哺乳類細胞中短暫表現諸抗體。使用標準蛋白A純化抗體,將其透析入PBS中供鑑定用。The antibodies are transiently expressed in mammalian cells by co-transfection of a suitable heavy and light chain containing a plastid. The antibody was purified using standard protein A and dialyzed into PBS for identification.
使用ELISA版式,與親代鼠類mAb C1068相較下,評估所有16個單株單體與人類TLR3(SEQ ID NO:4)胞外功能部位之結合作用。簡言之,將可溶性人類TLR3胞外功能部位塗覆於96槽盤之諸槽,於各種濃度(10- 3 至103 奈克/毫升)培育諸候選單株抗體,使用兔抗-小鼠IgG-HRP檢測鼠類IgG1同功型(Zymed,South San Francisco,CA)之結合抗體,或使用HRP-標記之抗-人類IgG(Jackson 109-036-088)檢測人類IgG4同功型之結合抗體。測定諸EC5 0 值,結果示於圖24及下文表7。Binding of all 16 monomeric monomers to the extracellular functional site of human TLR3 (SEQ ID NO: 4) was assessed using the ELISA format compared to the parental murine mAb C1068. Briefly, the soluble human TLR3 extracellular functional site was applied to the troughs of a 96-well plate, and the candidate monoclonal antibodies were incubated at various concentrations (10 - 3 to 10 3 Ng/ml) using rabbit anti-mouse. IgG-HRP detects binding antibodies to murine IgG1 isoforms (Zymed, South San Francisco, CA), or detects antibodies to human IgG4 isoforms using HRP-labeled anti-human IgG (Jackson 109-036-088) . EC 5 0 measured for various values of the results shown in FIG. 24 and Table 7 below.
C1068之EC5 0 計算值為8奈克/毫升;結果指示,相較於鼠類親代mAb 1068,12個適應人類單株抗體之EC5 0 計算值減少小於40倍。藉由測定利用Biacore之結合親和性及於細胞系細胞介素釋放分析試驗中之結合活性,進一步鑑定具有粗體字EC5 0 值之單株抗體。C1068 calculation of EC 50 value of 8 ng / mL; results indicate that, compared to the parental murine mAb 1068,12 adaptation of the human monoclonal antibody EC 50 of less than 40-fold reduction Calcd. By using the binding assay and Biacore affinity to cell lines of cytokine release assay Analysis of binding activity of monoclonal antibody having further characterized in bold EC 5 0 value.
利用Biacore之結合親和性測定係利用單株抗體捕獲技術及TLR3捕獲技術進行。單株抗體捕獲分析係於25℃使用備有CM5晶片[表面利用標準胺偶聯法,於25℃以蛋白A(6,000 RU)予以修飾]之Biacore 2000生物感應器進行。稀釋抗體至30 nM,於不同蛋白A表面捕獲一分鐘。注射0、0.1、0.3、1.0、3.0、及9.0 nM之TLR3,偵測結合與解離5分鐘。使用兩個6秒脈衝100 nM磷酸使經蛋白A修飾之表面再生。將可利用之結合數據組帶入1:1相互作用模式(CLAMPT M )中。計算速率常數與其比率(KD=kd /ka )及估算表觀平衡常數時帶來之適配誤差。Binding affinity assays using Biacore were performed using monoclonal antibody capture technology and TLR3 capture technology. The monoclonal antibody capture assay was performed at 25 ° C using a Biacore 2000 biosensor equipped with a CM5 wafer [surface modified with protein A (6,000 RU) at 25 ° C using a standard amine coupling method]. The antibody was diluted to 30 nM and captured on the surface of different Protein A for one minute. TLR3 at 0, 0.1, 0.3, 1.0, 3.0, and 9.0 nM was injected and binding and dissociation were detected for 5 minutes. Protein A modified surface was regenerated using two 6 second pulses of 100 nM phosphoric acid. The available binding data set is brought into the 1:1 interaction mode (CLAMP T M ). Calculate the adaptation error caused by the rate constant and its ratio (KD = kd / ka ) and the estimated apparent equilibrium constant.
TLR3捕獲分析係於25℃使用備有CM5晶片[表面利用標準胺偶聯法,於30℃以抗-His抗體(R&D Systems)(10,000 RU)予以修飾]之Biacore 3000生物感應器進行。於三個表面捕獲80、120、及300 RU密度之人類六-組織胺酸-TLR3,以第四個表面作為參照組。注射二重複之0、0.4、1.1、3.3、10、及30 nM抗體。偵測結合相三分鐘及偵測解離相七分鐘。使用兩個3-秒脈衝50 mM磷酸再生諸抗-His抗體表面。將可利用之結合數據組帶入經校正用於不同趨勢(drifts)之各單株抗體濃度概況之1:1相互作用模式(BIAevalT M )中。計算速率常數與其比率(KD=kd /ka )及估算表觀平衡常數時帶來之適配誤差。The TLR3 capture assay was performed at 25 ° C using a Biacore 3000 biosensor equipped with a CM5 wafer [surface modified with anti-His antibody (R&D Systems) (10,000 RU) at 30 °C using a standard amine coupling method]. Human hexa-histamine-TLR3 at a density of 80, 120, and 300 RU was captured on three surfaces with the fourth surface as the reference group. Two replicates of 0, 0.4, 1.1, 3.3, 10, and 30 nM antibodies were injected. The binding phase was detected for three minutes and the dissociation phase was detected for seven minutes. The anti-His antibody surface was regenerated using two 3-second pulses of 50 mM phosphoric acid. The available binding data sets were brought into a 1:1 interaction mode (BIAeval T M ) of individual antibody concentration profiles corrected for different trends (drifts). Calculate the adaptation error caused by the rate constant and its ratio (KD = kd / ka ) and the estimated apparent equilibrium constant.
經計算之KD 結果示於下文表8。兩個測定值代表1)晶片表面捕獲的抗-TLR3單株抗體與溶液中施加的人類TLR3之結合親和力及2)晶片表面捕獲的TLR3與施加於溶液相中的抗-TLR3單株抗體之結合親和力。結果指示,於利用固定化單株抗體捕獲溶液系TLR3時,所有候選單株抗體保持nM親和性,證實諸組合單株抗體已保留1068之結合特性。當TLR3於晶片上固定化時,多數候選單株抗體保留緊密結合特性,此結果與ELISA結合曲線一致。K D was calculated results are shown in Table 8 below it. The two measurements represent 1) the binding affinity of the anti-TLR3 monoclonal antibody captured on the surface of the wafer to the human TLR3 applied in the solution and 2) the binding of the TLR3 captured on the wafer surface to the anti-TLR3 monoclonal antibody applied to the solution phase. Affinity. The results indicated that all of the candidate monoclonal antibodies retained nM affinity when using the immobilized monoclonal antibody capture solution TLR3, confirming that the combined monoclonal antibodies retained the binding property of 1068. When TLR3 was immobilized on the wafer, most of the candidate monoclonal antibodies retained tight binding properties, and this result was consistent with the ELISA binding curve.
利用Biacore測定之適應人類抗-TLR3單株抗體之結合活性亦於細胞系細胞介素釋放分析試驗中予以測定。將人類肺上皮細胞株BEAS-2B塗佈於96槽盤,於細胞中添加多(I:C)或與候選抗體於不含血清基質中預培育之多(I:C)。4天後,移出條件式培養基,利用Luminex技術測定可溶性細胞介素含量。結果示於圖25,證明適應人類之單株抗體保持親代mAb C1068之生物活性,亦即,由使用TLR3配位體多(I:C)攻毒之細胞促炎性細胞介素產生減少測得之TLR3活性中和作用。The binding activity of the human anti-TLR3 monoclonal antibody assay determined by Biacore was also determined in a cell line interleukin release assay. The human lung epithelial cell line BEAS-2B was plated on a 96-well plate, and multiple (I:C) was added to the cells or pre-incubated with the candidate antibody in a serum-free matrix (I:C). After 4 days, remove the conditioned medium and use Luminex The technique measures the soluble interleukin content. The results are shown in Figure 25, demonstrating that the individual antibodies adapted to humans retain the biological activity of the parental mAb C1068, i.e., the reduction of pro-inflammatory cytokine production by cells challenged with TLR3 ligands (I:C). The neutralizing effect of TLR3 activity was obtained.
適應人類之 C1068 重鏈與輕鏈變異體之產生及鑑定 鼠類抗-TLR3 mAb 1068 CDRs之電腦篩選免疫原性分析揭示可操作以減少序列之免疫原性計分之於CDR界限內之一系列聚集部位(aggretopes)。一旦鑑定出可予以操作之區域,則應用序列以及結構準則以決定必須使用之胺基酸替換。使用彼等準則,於重鏈可變區(VH )鑑定出四個單一點-胺基酸替換;於輕鏈可變區(V)鑑定出三個突變(單一突變、雙突變及三突變)。所有八個突變於HV1/LV1背景中互相獨立地進行,並列於表9。同時應用另一種替換,以決定M102殘基轉變為異白胺酸之效應,完成CDRs中全數甲硫胺酸之轉換,因為彼等殘基於轉譯後會被氧化,一種可能對蛋白質溶解性有害之修飾作用。如上文所述產生彼等抗體,並評估TLR3結合作用(見表10與11)及生物活性(見圖26-30)。 Generation and Identification of C1068 Heavy and Light Chain Variants Adapted to Humans Computer Screening for Murine Anti-TLR3 mAb 1068 CDRs Immunogenicity analysis revealed a series of CDR boundaries operable to reduce the immunogenicity of the sequence Aggregates (aggretopes). Once the area that can be manipulated is identified, the sequence and structural criteria are applied to determine the amino acid substitution that must be used. Using the criteria, four single point-amino acid substitutions were identified in the heavy chain variable region ( VH ); in the light chain variable region (V) Three mutations (single, double and triple) were identified. All eight mutations were performed independently of each other in the HV1/LV1 background and are listed in Table 9. At the same time, another substitution is applied to determine the effect of the conversion of the M102 residue to isoleucine, completing the conversion of all methionine in the CDRs, since the residues are oxidized based on translation, one may be harmful to protein solubility. Modification. Their antibodies were generated as described above and assessed for TLR3 binding (see Tables 10 and 11) and biological activity (see Figures 26-30).
如對抗人類TLR3之結合EC5 0 所示,嫁接於HV1/LV1背景內之1068 CDRs Vh中進行之所有五個單一點突變均具耐性。HV1/LV1背景測得之EC5 0 為29.2奈克/毫升;134M與Y60G二者之EC5 0 值均較低,分別為17與14.6奈克/毫升。此暗示彼等變化不僅減少HV1/LV1之電腦篩選免疫原性,亦增進與TLR3之結合作用。其他三個突變之結合作用比HV1/LV1稍微弱些。All five single point mutations such as against human TLR3 binding of EC, the grafted 1068 CDRs Vh within HV1 / LV1 background 50 are performed with the resistance. HV1 / LV1 measured background of EC 5 0 to 29.2 ng / mL; 134M and between the two Y60G EC 5 0 values are lower, respectively, 17 and 14.6 ng / ml. This suggests that these changes not only reduce the computerized immunogenicity of HV1/LV1 but also enhance the binding to TLR3. The combination of the other three mutations is slightly weaker than HV1/LV1.
V1之CDR1中之突變無一者具耐性(EC5 0 >1000奈克/毫升),暗示此區域對於1068如何識別人類TLR3具決定性。None of the mutations in CDR1 of V1 were resistant (EC 5 0 >1000 Ng/ml), suggesting that this region is decisive for how 1068 recognizes human TLR3.
頃已完整敘述本發明,對於熟習此項技藝人士而言,在不偏離隨附專利申請之精神或範圍下之許多變化及修飾乃顯見之事。The present invention has been fully described, and it is obvious to those skilled in the art that many changes and modifications can be made without departing from the spirit or scope of the appended claims.
圖1顯示得自人類TLR3(hTLR3)單株抗體拮抗劑之重鏈可變區序列(CDRs劃出底線)。Figure 1 shows the heavy chain variable region sequences (CDRs bottom line) from human TLR3 (hTLR3) monoclonal antibody antagonists.
圖2顯示得自hTLR3單株抗體拮抗劑之輕鏈可變區序列(CDRs劃出底線)。Figure 2 shows the light chain variable region sequences (CDRs from the bottom line) obtained from hTLR3 monoclonal antibody antagonists.
圖3顯示利用TLR3拮抗劑於人類肺上皮衍生細胞中抑制由多(I:C)誘發之IL-6細胞介素產生。Figure 3 shows inhibition of IL-6 interleukin production induced by poly(I:C) in human lung epithelial-derived cells using a TLR3 antagonist.
圖4顯示利用TLR3拮抗劑於人類肺上皮衍生細胞中抑制由多(I:C)誘發之IL-8細胞介素產生。Figure 4 shows inhibition of IL-8 interleukin production induced by poly(I:C) in human lung epithelial-derived cells using a TLR3 antagonist.
圖5顯示利用TLR3拮抗劑於人類肺上皮衍生細胞中抑制由多(I:C)誘發之RANTES細胞介素產生。Figure 5 shows inhibition of RANTES interleukin production induced by poly(I:C) in human lung epithelial-derived cells using a TLR3 antagonist.
圖6顯示利用TLR3拮抗劑於初級人類支氣管-上皮細胞中抑制由多(I:C)誘發之MIP1-α 細胞介素產生。Figure 6 shows inhibition of MIP1- alpha interleukin production induced by poly(I:C) in primary human bronchial-epithelial cells using a TLR3 antagonist.
圖7顯示利用TLR3拮抗劑於初級人類支氣管-上皮細胞中抑制由多(I:C)誘發之IL-6細胞介素產生。Figure 7 shows inhibition of IL-6 interleukin production induced by poly(I:C) in primary human bronchial-epithelial cells using a TLR3 antagonist.
圖8顯示剔除TLR3活性對與IBD-相關的減重之影響。Figure 8 shows the effect of knockout TLR3 activity on IBD-related weight loss.
圖9顯示利用TLR3拮抗劑抑制與IBD-相關的減重。Figure 9 shows inhibition of IBD-related weight loss with TLR3 antagonists.
圖10顯示經由以TLR3拮抗劑處理,鼠類敗血症模式中存活率增加。Figure 10 shows an increase in survival in a murine sepsis mode via treatment with a TLR3 antagonist.
圖11顯示利用TLR3拮抗劑,鼠類敗血症模式中IL-6細胞介素生產降低。Figure 11 shows the reduction in IL-6 interleukin production in a murine sepsis model using a TLR3 antagonist.
圖12顯示利用TLR3拮抗劑,鼠類敗血症模式中TNF-α細胞介素生產降低。Figure 12 shows a reduction in TNF-[alpha] interleukin production in a murine sepsis model using a TLR3 antagonist.
圖13顯示於鼠類肺組織中,由多(I:C)誘發的炎性細胞總數增加。Figure 13 shows an increase in the total number of inflammatory cells induced by multiple (I:C) in murine lung tissue.
圖14顯示於鼠類肺組織中,由多(I:C)誘發的嗜中性白血球增加。Figure 14 shows an increase in neutrophils induced by multiple (I:C) in murine lung tissue.
圖15顯示於鼠類肺組織中,由多(I:C)誘發的單核炎性細胞增加。Figure 15 shows an increase in mononuclear inflammatory cells induced by multiple (I:C) in murine lung tissue.
圖16顯示使用單一劑量多(I:C)之TLR3活化,進一步於受乙醯甲基膽鹼攻毒之小鼠中傷害肺功能。Figure 16 shows the use of a single dose of multiple (I:C) TLR3 activation to further impair lung function in mice challenged with acetaminophen.
圖17顯示使用多劑量多(I:C)之TLR3活化,進一步於受乙醯甲基膽鹼攻毒之小鼠中傷害肺功能。Figure 17 shows the use of multiple doses of multiple (I:C) TLR3 activation to further impair lung function in mice challenged with ethylene methylcholine.
圖18顯示TLR3基因剔除小鼠,於乙醯甲基膽鹼攻毒期間,免受由單一劑量多(I:C)誘發的肺功能傷害。Figure 18 shows TLR3 knockout mice protected from a single dose of multiple (I:C)-induced lung function damage during challenge with methotrexate methylcholine.
圖19顯示TLR3基因剔除小鼠,於乙醯甲基膽鹼攻毒期間,免受由多劑量多(I:C)誘發的肺功能傷害。Figure 19 shows TLR3 knockout mice protected from multi-dose (I:C)-induced lung function damage during challenge with methotrexate methylcholine.
圖20顯示TLR3拮抗劑對人類肺支氣管上皮細胞中細胞介素與化學激素產生之影響。Figure 20 shows the effect of TLR3 antagonists on the production of interleukins and chemical hormones in human lung bronchial epithelial cells.
圖21顯示經由使用TLR3拮抗劑預防及治療,鼠類致命性肺炎模式中存活率增加。Figure 21 shows an increase in survival in a murine lethal pneumonia pattern by prophylaxis and treatment with a TLR3 antagonist.
圖22顯示經未達致死劑量之流行性感冒病毒A/PR/8與肺炎鏈球菌(Streptococcus pneumoniae )感染後,於鼠類模式中致命性肺炎之進展。Figure 22 shows the progression of fatal pneumonia in a murine model after infection with influenza A virus A/PR/8 and Streptococcus pneumoniae .
圖23顯示經流行性感冒病毒A/PR/8與肺炎鏈球菌感染之小鼠肺中之細菌負擔。Figure 23 shows the bacterial burden in the lungs of mice infected with influenza virus A/PR/8 and S. pneumoniae.
圖24A、B、C與D顯示於ELISA分析試驗中,適應人類之抗-TLR3單株抗體與hTLR3之結合作用。Figures 24A, B, C and D show the binding of human anti-TLR3 monoclonal antibody to hTLR3 in an ELISA assay.
圖25顯示於細胞系細胞介素釋放分析試驗中,適應人類之抗-TLR3單株抗體之評估。Figure 25 shows the evaluation of human anti-TLR3 monoclonal antibodies in a cell line interleukin release assay.
圖26顯示於使用IP-10讀出裝置之細胞系生物活性分析試驗中,變異單株抗體HBV1至HBV8(HBV4除外)之評估。Figure 26 shows the evaluation of variant monoclonal antibodies HBV1 to HBV8 (except HBV4) in a cell line biological activity assay using an IP-10 readout.
圖27顯示於使用RANTES讀出裝置之細胞系生物活性分析試驗中,變異單株抗體HBV1至HBV8(HBV4除外)之評估。Figure 27 shows the evaluation of variant monoclonal antibodies HBV1 to HBV8 (except HBV4) in a cell line biological activity assay using a RANTES readout.
圖28顯示於使用IL-8讀出裝置之細胞系生物活性分析試驗中,變異單株抗體HBV1至HBV8(HBV4除外)之評估。。Figure 28 shows the evaluation of variant monoclonal antibodies HBV1 to HBV8 (except HBV4) in a cell line biological activity assay using an IL-8 readout. .
圖29顯示於使用MCP-1讀出裝置之細胞系生物活性分析試驗中,變異單株抗體HBV1至HBV8(HBV4除外)之評估。Figure 29 shows the evaluation of variant monoclonal antibodies HBV1 to HBV8 (except HBV4) in a cell line biological activity assay using the MCP-1 readout.
圖30顯示於使用IL-6讀出裝置之細胞系生物活性分析試驗中,變異單株抗體HBV1至HBV8(HBV4除外)之評估。Figure 30 shows the evaluation of variant monoclonal antibodies HBV1 to HBV8 (except HBV4) in a cell line biological activity assay using an IL-6 readout.
圖31A及圖31B顯示高脂飼料餵飼14週(A)及26週(B)之TLR3基因剔除小鼠防止與高脂餵飼相關的葡萄糖耐受不良之進展。Figure 31A and Figure 31B show that TLR3 knockout mice fed a high fat diet for 14 weeks (A) and 26 weeks (B) prevented progression of glucose intolerance associated with high fat feeding.
圖32顯示TLR3基因剔除小鼠高脂餵飼26週後,具有正常之空腹血糖量。Figure 32 shows that TLR3 knockout mice have normal fasting blood glucose levels after 26 weeks of high fat feeding.
圖33A、B與C顯示以高脂飼料餵飼TLR3基因剔除小鼠26週,於基線(未進行葡萄糖攻毒)(圖33A)、經葡萄糖攻毒後20分鐘後(圖33B)與60分鐘後(圖33C),空腹胰島素含量增加。Figures 33A, B and C show TLR3 knockout mice fed a high fat diet for 26 weeks at baseline (no glucose challenge) (Figure 33A), 20 minutes after glucose challenge (Figure 33B) and 60 minutes After (Fig. 33C), the fasting insulin content increased.
圖34A、B、C、D與E顯示經高脂餵飼30週後,相較於以高脂飼料餵飼之野生型小鼠,TLR3基因剔除小鼠之脂質概況獲改善,如總膽固醇(圖34A)、HDL(圖34B)、LDL(圖34C)、FFA(圖34D)及HDLc/LDLc比率(圖34E)。圖35顯示於誘發慢性DSS結腸炎期間,使用TLR3拮抗劑之預防(Pr)與治療(T)處理。Figures 34A, B, C, D, and E show that after 30 weeks of high-fat feeding, the lipid profile of TLR3 knockout mice was improved, such as total cholesterol, compared to wild-type mice fed a high-fat diet. Figure 34A), HDL (Figure 34B), LDL (Figure 34C), FFA (Figure 34D), and HDLc/LDLc ratios (Figure 34E). Figure 35 shows prophylactic (Pr) and therapeutic (T) treatments with TLR3 antagonists during induction of chronic DSS colitis.
圖36顯示各DSS攝取週期出現之減重。Figure 36 shows the weight loss that occurs for each DSS uptake cycle.
圖37顯示第二DSS週期後之減重及回復。Figure 37 shows the weight loss and recovery after the second DSS cycle.
圖38顯示第三DSS週期後之減重及回復。Figure 38 shows the weight loss and recovery after the third DSS cycle.
圖39顯示TLR3拮抗劑處理對與慢性DSS結腸炎相關之淨減重之影響。Figure 39 shows the effect of TLR3 antagonist treatment on net weight loss associated with chronic DSS colitis.
圖40顯示TLR3拮抗劑處理對與慢性DSS結腸炎相關之結腸變短之影響。Figure 40 shows the effect of TLR3 antagonist treatment on colon shortening associated with chronic DSS colitis.
圖41A、B與C顯示TLR3拮抗劑處理對慢性DSS結腸炎嚴重性之影響。圖41A為各組別之總和;圖41B為各組溫和組織病理學變化;圖41C為慢性修復性組織病理學變化。圖41D、E與F顯示hTLR3拮抗劑處理於慢性DSS結腸炎中之組織病理作用。圖41D為各組別之上皮潰瘍形成;圖41E為各組別之固有層肉芽組織之形成;圖41F為各組別之黏膜下層肉芽組織之形成。Figures 41A, B and C show the effect of TLR3 antagonist treatment on the severity of chronic DSS colitis. Figure 41A is the sum of the groups; Figure 41B is the mild histopathological changes of each group; Figure 41C is the chronic repair histopathological changes. Figures 41D, E and F show the histopathological effects of hTLR3 antagonist treatment in chronic DSS colitis. Fig. 41D shows the formation of epithelial ulcers in each group; Fig. 41E shows the formation of the granulation tissue of the lamina propria of each group; Fig. 41F shows the formation of the granulation tissue of the submucosa of each group.
圖42顯示慢性DSS結腸炎中之T-細胞活化作用。Figure 42 shows T-cell activation in chronic DSS colitis.
圖43顯示預防性TLR3拮抗劑處理對脾臟中與DSS相關之CD11b+細胞增加之影響。Figure 43 shows the effect of prophylactic TLR3 antagonist treatment on the increase in DSS-associated CD11b+ cells in the spleen.
圖44顯示TLR3拮抗劑處理對慢性DSS結腸炎中IL-4與IL-10全身含量之影響。Figure 44 shows the effect of TLR3 antagonist treatment on systemic levels of IL-4 and IL-10 in chronic DSS colitis.
<110> 卡爾頓/CARTON,JILL M. 陳欣柔/CHEN,SHIZHONG 邱寧翰/CUNNINGHAM,MARK 達安克/DAS,ANUK 杜凱倫/DUFFY,KAREN 吉雷斯/GILES-KOMAR,JILL M. 高磊茲/GOLETZ,THERESA J. 奈大衛/KNIGHT,DAVID 羅柏塔/LAMB,ROBERTA 摩哈瑪/MBOW,M.LAMINE 克莉絲/PICHA,KRISTEN 拉谷翰/RAGHUNATHAN,GOPALAN 馬特歐/SAN MATEO,LANI 薩黎斯/SARISKY,ROBERT T. 史托維/STOWELL,NICOLE 史維克/STOJANOVIC-SUSULIC,VEDRANA 史雷門/SWEET,RAYMOND 趙善榮/ZHAO,SHANRONG <120> 類鐸受體3拮抗劑,方法及用途<130> CEN5083 USA NP <140> 待指定<141> 2005-11-30 <150> 60/631,815 <151> 2004-11-30 <150> 60/636,399 <151> 2004-12-15 <150> 60/641,877 <151> 2005-01-06 <150> 60/713,195 <151> 2005-08-31 <150> 60/727,610 <151> 2005-10-18 <160> 63 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 2712 <212> DNA <213> 智人<400> 1 <210> 3 <211> 2109 <212> DNA <213> 智人<400> 3<210> 4 <211> 703 <212> PRT <213> 智人<400> 4 <210> 5 <211> 381 <212> DNA <213> 小家鼠<400> 5<210> 6 <211> 138 <212> PRT <213> 小家鼠<400> 6 <210> 7 <211> 19 <212> PRT <213> 小家鼠<400> 7<210> 8 <211> 29 <212> PRT <213> 小家鼠<400> 8<210> 9 <211> 6 <212> PRT <213> 小家鼠<400> 9<210> 10 <211> 14 <212> PRT <213> 小家鼠<400> 10<210> 11 <211> 17 <212> PRT <213> 小家鼠<400> 11 <210> 12 <211> 32 <212> PRT <213> Mus musculus <400> 12<210> 13 <211> 10 <212> PRT <213> 小家鼠<400> 13<210> 14 <211> 11 <212> PRT <213> 小家鼠<400> 14<210> 15 <211> 381 <212> DNA <213> 小家鼠<400> 15<210> 16 <211> 127 <212> PRT <213> 小家鼠<400> 16 <210> 17 <211> 20 <212> PRT <213> 小家鼠<400> 17<210> 18 <211> 23 <212> PRT <213> 小家鼠<400> 18<210> 19 <211> 11 <212> PRT <213> 小家鼠<400> 19<210> 20 <211> 15 <212> PRT <213> 小家鼠<400> 20<210> 21 <211> 7 <212> PRT <213> 小家鼠<400> 21<210> 22 <211> 32 <212> PRT <213> 小家鼠<400> 22<210> 23 <211> 7 <212> PRT <213> 小家鼠<400> 23<210> 24 <211> 12 <212> PRT <213> 小家鼠<400> 24<210> 25 <211> 119 <212> PRT <213> 人工序列<220> <223> 適應人類之重鏈HV1 <400> 25 <210> 26 <211> 357 <212> DNA <213> 人工序列<220> <223> 適應人類之重鏈HV1 <400> 26<210> 27 <211> 119 <212> PRT <213> 人工序列<220> <223> 適應人類之重鏈HV4 <400> 27<210> 28 <211> 357 <212> DNA <213> 人工序列<220> <223> 適應人類之重鏈HV4 <400> 28<210> 29 <211> 119 <212> PRT <213> 人工序列<220> <223> 適應人類之重鏈HV5 <400> 29<210> 30 <211> 357 <212> DNA <213> 人工序列<220> <223> 適應人類之重鏈HV5 <400> 30 <210> 31 <211> 119 <212> PRT <213> 人工序列<220> <223> 適應人類之重鏈HV7 <400> 31<210> 32 <211> 357 <212> DNA <213> 人工序列<220> <223> 適應人類之重鏈HV7 <400> 32<210> 33 <211> 107 <212> PRT <213> 人工序列<220> <223> 適應人類之輕鏈LV1 <400> 33 <210> 34 <211> 321 <212> DNA <213> 人工序列<220> <223> 適應人類之輕鏈Lv1 <400> 34<210> 35 <211> 107 <212> PRT <213> 人工序列<220> <223> 適應人類之輕鏈LV3 <400> 35<210> 36 <211> 321 <212> DNA <213> 人工序列<220> <223> 適應人類之輕鏈LV3 <400> 36<210> 37 <211> 107 <212> PRT <213> 人工序列<220> <223> 適應人類之輕鏈LV5 <400> 37<210> 38 <211> 321 <212> DNA <213> 人工序列<220> <223> 適應人類之輕鏈LV5 <400> 38<210> 39 <211> 107 <212> PRT <213> 人工序列<220> <223> 適應人類之輕鏈LV7 <400> 39<210> 40 <211> 321 <212> DNA <213> 人工序列<220> <223> 適應人類之輕鏈LV7 <400> 40<210> 41 <211> 327 <212> PRT <213> 人工序列<220> <223> 人類IgG4重鏈恒定區變異體<400> 41 <210> 42 <211> 981 <212> DNA <213> 人工序列<220> <223> 人類IgG4重鏈恒定區變異體<400> 42 <210> 43 <211> 107 <212> PRT <213> 人工序列<220> <223> 人類k恒定區<400> 43<210> 44 <211> 324 <212> DNA <213> 人工序列<220> <223> 人類k恒定區<400> 44<210> 45 <211> 119 <212> PRT <213> 人工序列<220> <223> 適應人類之重鏈變異體HBV1 <400> 45 <210> 46 <211> 357 <212> DNA <213> 人工序列<220> <223> 適應人類之重鏈變異體HBV1 <400> 46<210> 47 <211> 119 <212> PRT <213> 人工序列<220> <223> 適應人類之重鏈變異體HBV2 <400> 47<210> 48 <211> 357 <212> DNA <213> 人工序列<220> <223> 適應人類之重鏈變異體HBV2 <400> 48<210> 49 <211> 119 <212> PRT <213> 人工序列<220> <223> 適應人類之重鏈變異體HBV3 <400> 49<210> 50 <211> 357 <212> DNA <213> 人工序列<220> <223> 適應人類之重鏈變異體HBV3 <400> 50 <210> 51 <211> 119 <212> PRT <213> 人工序列<220> <223> 適應人類之重鏈變異體HBV4 <400> 51<210> 52 <211> 357 <212> DNA <213> 人工序列<220> <223> 適應人類之重鏈變異體HBV4 <400> 52<210> 53 <211> 119 <212> PRT <213> 人工序列<220> <223> 適應人類之重鏈變異體HBV5 <400> 53<210> 54 <211> 357 <212> DNA <213> 人工序列<220> <223> 適應人類之重鏈變異體HBV5 <400> 54<210> 55 <211> 107 <212> PRT <213> 人工序列<220> <223> 適應人類之輕鏈變異體HBV6 <400> 55<210> 56 <211> 321 <212> DNA <213> 人工序列<220> <223> 適應人類之輕鏈變異體HBV6 <400> 56<210> 57 <211> 107 <212> PRT <213> 人工序列<220> <223> 適應人類之輕鏈變異體HBV7 <400> 57<210> 58 <211> 321 <212> DNA <213> 人工序列<220> <223> 適應人類之輕鏈變異體HBV7 <400> 58 <210> 59 <211> 107 <212> PRT <213> 人工序列<220> <223> 適應人類之輕鏈變異體HBV8 <400> 59<210> 60 <211> 321 <212> DNA <213> 人工序列<220> <223> 適應人類之輕鏈變異體HBV8 <400> 60<210> 61 <211> 6 <212> PRT <213> 人工序列<220> <221> 不確定<222> (5) <223> 重鏈CDR1其中位置5之Xaa可為異白胺酸或甲硫胺酸<400> 61 <210> 62 <211> 17 <212> PRT <213> 人工序列<220> <221> 不確定<222> (11) <223> 重鏈CDR2其中位置11之Xaa可為酪胺酸或甘胺酸<220> <221> 不確定<222> (12) <223> 重鏈CDR2其中位置12之Xaa可為天冬醯胺或丙胺酸<220> <221> 不確定<222> (15) <223> 重鏈CDR2其中位置15之Xaa可為苯丙胺酸或甘胺酸<400> 62<210> 63 <211> 10 <212> PRT <213> 人工序列<220> <221> 不確定<222> (4) <223> 重鏈CDR3其中位置4之Xaa可為甲硫胺酸或異白胺酸<400> 63 <110> Carlton/CARTON, JILL M. Chen Xinrou/CHEN, SHIZHONG Qiu Ninghan/CUNNINGHAM, MARK Dianke/DAS, ANUK Dukalun/DUFFY, KAREN Gilles/GILES-KOMAR, JILL M. Gao Leizi/ GOLETZ,THERESA J. Nai David/KNIGHT, DAVID Roberta/LAMB, ROBERTA Mohama/MBOW, M.LAMINE KRIS/PICHA, KRISTEN Rakuhan/RAGHUNATHAN, GOPALAN Mateo/SAN MATEO, LANI SARIS/SARISKY, ROBERT T. STOWELL/STOWELL, NICOLE Svik/STOJANOVIC-SUSULIC, VEDRANA SWEET/SWEET, RAYMOND ZHAO Shanrong/ZHAO, SHARONG <120> 铎 receptor 3 antagonist, method and use <130> CEN5083 USA NP <140> To be specified <141> 2005-11-30 <150> 60/631,815 <151> 2004-11-30 <150> 60/636,399 <151> 2004-12-15 <150> 60/641,877 <151> 2005-01-06 <150> 60/713,195 <151> 2005-08-31 <150> 60/727,610 <151> 2005-10-18 <160> 63 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 2712 <212> DNA <213> Homo sapiens <400> 1 <210> 3 <211> 2109 <212> DNA <213> Homo sapiens <400> 3 <210> 4 <211> 703 <212> PRT <213> Homo sapiens <400> 4 <210> 5 <211> 381 <212> DNA <213> Mus musculus <400> 5 <210> 6 <211> 138 <212> PRT <213> Mus musculus <400> 6 <210> 7 <211> 19 <212> PRT <213> Mus musculus <400> 7 <210> 8 <211> 29 <212> PRT <213> Mus musculus <400> 8 <210> 9 <211> 6 <212> PRT <213> Mus musculus <400> 9 <210> 10 <211> 14 <212> PRT <213> Mus musculus <400> 10 <210> 11 <211> 17 <212> PRT <213> Mus musculus <400> 11 <210> 12 <211> 32 <212> PRT <213> Mus musculus <400> 12 <210> 13 <211> 10 <212> PRT <213> Mus musculus <400> 13 <210> 14 <211> 11 <212> PRT <213> Mus musculus <400> 14 <210> 15 <211> 381 <212> DNA <213> Mus musculus <400> 15 <210> 16 <211> 127 <212> PRT <213> Mus musculus <400> 16 <210> 17 <211> 20 <212> PRT <213> Mus musculus <400> 17 <210> 18 <211> 23 <212> PRT <213> Mus musculus <400> 18 <210> 19 <211> 11 <212> PRT <213> Mus musculus <400> 19 <210> 20 <211> 15 <212> PRT <213> Mus musculus <400> 20 <210> 21 <211> 7 <212> PRT <213> Mus musculus <400> 21 <210> 22 <211> 32 <212> PRT <213> Mus musculus <400> 22 <210> 23 <211> 7 <212> PRT <213> Mus musculus <400> 23 <210> 24 <211> 12 <212> PRT <213> Mus musculus <400> 24 <210> 25 <211> 119 <212> PRT <213> Artificial sequence <220><223> Adapted to human heavy chain HV1 <400> 25 <210> 26 <211> 357 <212> DNA <213> Artificial sequence <220><223> Adapted to human heavy chain HV1 <400> 26 <210> 27 <211> 119 <212> PRT <213> Artificial sequence <220><223> Adaptation to human heavy chain HV4 <400> 27 <210> 28 <211> 357 <212> DNA <213> Artificial sequence <220><223> Adapted to human heavy chain HV4 <400> 28 <210> 29 <211> 119 <212> PRT <213> Artificial sequence <220><223> Adapted to human heavy chain HV5 <400> 29 <210> 30 <211> 357 <212> DNA <213> Artificial sequence <220><223> Adapted to human heavy chain HV5 <400> 30 <210> 31 <211> 119 <212> PRT <213> Artificial sequence <220><223> Adapted to human heavy chain HV7 <400> 31 <210> 32 <211> 357 <212> DNA <213> Artificial sequence <220><223> Adapted to human heavy chain HV7 <400> 32 <210> 33 <211> 107 <212> PRT <213> Artificial sequence <220><223> Adapted to human light chain LV1 <400> 33 <210> 34 <211> 321 <212> DNA <213> Artificial sequence <220><223> Adapted to human light chain Lv1 <400> 34 <210> 35 <211> 107 <212> PRT <213> Artificial sequence <220><223> Adapted to human light chain LV3 <400> 35 <210> 36 <211> 321 <212> DNA <213> Artificial sequence <220><223> Adapted to human light chain LV3 <400> 36 <210> 37 <211> 107 <212> PRT <213> Artificial sequence <220><223> Adapted to human light chain LV5 <400> 37 <210> 38 <211> 321 <212> DNA <213> Artificial sequence <220><223> Adapted to human light chain LV5 <400> 38 <210> 39 <211> 107 <212> PRT <213> Artificial sequence <220><223> Adapted to human light chain LV7 <400> 39 <210> 40 <211> 321 <212> DNA <213> Artificial sequence <220><223> Adapted to human light chain LV7 <400> 40 <210> 41 <211> 327 <212> PRT <213> Artificial sequence <220><223> Human IgG4 heavy chain constant region variant <400> 41 <210> 42 <211> 981 <212> DNA <213> Artificial sequence <220><223> Human IgG4 heavy chain constant region variant <400> 42 <210> 43 <211> 107 <212> PRT <213> Artificial sequence <220><223> Human k constant region <400> 43 <210> 44 <211> 324 <212> DNA <213> Artificial sequence <220><223> Human k constant region <400> 44 <210> 45 <211> 119 <212> PRT <213> Artificial sequence <220><223> Adaptation to human heavy chain variant HBV1 <400> 45 <210> 46 <211> 357 <212> DNA <213> Artificial sequence <220><223> Adaptation to human heavy chain variant HBV1 <400> 46 <210> 47 <211> 119 <212> PRT <213> Artificial sequence <220><223> Adaptation to human heavy chain variant HBV2 <400> 47 <210> 48 <211> 357 <212> DNA <213> Artificial sequence <220><223> Adaptation to human heavy chain variant HBV2 <400> 48 <210> 49 <211> 119 <212> PRT <213> Artificial sequence <220><223> Adapted to human heavy chain variant HBV3 <400> 49 <210> 50 <211> 357 <212> DNA <213> Artificial sequence <220><223> Adaptation to human heavy chain variant HBV3 <400> 50 <210> 51 <211> 119 <212> PRT <213> Artificial sequence <220><223> Adaptation to human heavy chain variant HBV4 <400> 51 <210> 52 <211> 357 <212> DNA <213> Artificial sequence <220><223> Adaptation to human heavy chain variant HBV4 <400> 52 <210> 53 <211> 119 <212> PRT <213> Artificial sequence <220><223> Adaptation to human heavy chain variant HBV5 <400> 53 <210> 54 <211> 357 <212> DNA <213> Artificial sequence <220><223> Adaptation to human heavy chain variant HBV5 <400> 54 <210> 55 <211> 107 <212> PRT <213> Artificial sequence <220><223> Adapted to human light chain variant HBV6 <400> 55 <210> 56 <211> 321 <212> DNA <213> Artificial sequence <220><223> Adapted to human light chain variant HBV6 <400> 56 <210> 57 <211> 107 <212> PRT <213> Artificial sequence <220><223> Adapted to human light chain variant HBV7 <400> 57 <210> 58 <211> 321 <212> DNA <213> Artificial sequence <220><223> Adaptation to human light chain variant HBV7 <400> 58 <210> 59 <211> 107 <212> PRT <213> Artificial sequence <220><223> Adapted to human light chain variant HBV8 <400> 59 <210> 60 <211> 321 <212> DNA <213> Artificial sequence <220><223> Adapted to human light chain variant HBV8 <400> 60 <210> 61 <211> 6 <212> PRT <213> Artificial sequence <220><221> Uncertain <222> (5) <223> Heavy chain CDR1 where Xaa at position 5 may be iso-leucine or A Thiamine <400> 61 <210> 62 <211> 17 <212> PRT <213> Artificial sequence <220><221> Uncertain <222> (11) <223> Heavy chain CDR2 where Xaa at position 11 can be tyrosine or glycine Acid <220><221> Not determined <222> (12) <223> Heavy chain CDR2 where Xaa at position 12 can be aspartame or alanine <220><221> Not determined <222> (15) <223> Heavy chain CDR2 wherein Xaa at position 15 can be phenylalanine or glycine <400> 62 <210> 63 <211> 10 <212> PRT <213> Artificial sequence <220><221> Uncertain <222> (4) <223> Heavy chain CDR3 where Xaa at position 4 may be methionine or different Leucine <400> 63
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EP1824514A2 (en) | 2007-08-29 |
HK1131744A1 (en) | 2010-02-05 |
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BRPI0518664A2 (en) | 2008-12-02 |
CA2589636A1 (en) | 2006-06-08 |
HK1111360A1 (en) | 2008-08-08 |
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CA2589636C (en) | 2015-03-24 |
IL183541A (en) | 2011-10-31 |
US20060115475A1 (en) | 2006-06-01 |
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WO2006060513A2 (en) | 2006-06-08 |
KR101317264B1 (en) | 2013-10-15 |
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US8153583B2 (en) | 2012-04-10 |
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